Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Wang, Han
Doctor of Philosophy - PhD
University of British Columbia
Understanding the molecular mechanism of protein folding has always remained a challenging problem. Extensive investigations based on ensemble methods have been carried out to shed light on the protein folding problem. Compared with ensemble-averaged measurements, single-molecule force spectroscopy (SMFS) has evolved as a powerful technique to probe mechanical unfolding/folding of proteins at the single-molecule level, which provides rich details of protein conformational changes. Optical tweezers (OT), as one representative SMFS technique, have been widely used to directly monitor a protein folding process. This thesis presents a series of studies that use OT to investigate the mechanical unfolding/folding of two distinct classes of proteins: knotted/slipknotted proteins and Ca²⁺-binding metalloproteins. First, we combined OT and steered molecular dynamics (SMD) simulations to investigate a complex slipknotted protein. When stretched from the N- and C-termini, the slipknotted structure could be completely untied via multiple unfolding pathways. Upon relaxation, this protein showed complex folding behaviors involving misfolding. Our studies demonstrate the unfolding/folding mechanisms of a slipknotted protein with a complex topological conformation. Second, we used OT to mechanically tighten, untie and retie a trefoil knotted protein. Our results revealed that protein folding with a preformed knot was fast and robust; however, the folding from the unfolded polypeptide chain without the knot conformation was significantly slower, suggesting that the knotting was the rate-limiting step for the folding of this trefoil knotted protein. Next, we investigated the mechanical unfolding/folding behaviors of a Ca²⁺-binding protein, RTX (repeats-in-toxin) block V. Our results elucidated the secretion mechanism of this RTX block V and revealed that the mechanical design can ensure an efficient translocation process. Finally, we used OT to study another Ca²⁺-binding protein, RTX block IV. The mechanical properties of RTX block IV was well-characterized. Our results revealed that RTX block IV follows a similar translocation mechanism to be secreted as RTX block V. We also suggested that the secreted RTX block V is stabilized by folded RTX block IV and protected from being unfolded in vivo. Overall, this thesis clarified the unfolding/folding mechanisms of two knotted/slipknotted proteins and Ca²⁺-binding protein RTX.
INVESTIGATING FORCE-INDUCED UNFOLDING AND FOLDING OF PROTEINS AT THE SINGLE MOLECULE LEVEL by Han Wang B.Sc., Wuhan University, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2021 © Han Wang, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Investigating force-induced unfolding and folding of proteins at the single molecule level submitted by Han Wang in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Examining Committee: Hongbin Li, Chemistry, UBC Supervisor Suzana K Straus, Chemistry, UBC Supervisory Committee Member Russ Algar, Chemistry, UBC University Examiner Roman Krems, Chemistry, UBC University Examiner Additional Supervisory Committee Members: Katherine S. Ryan, Chemistry, UBC Supervisory Committee Member Derek P Gates, Chemistry, UBC Supervisory Committee Member iii Abstract Understanding the molecular mechanism of protein folding has always remained a challenging problem. Extensive investigations based on ensemble methods have been carried out to shed light on the protein folding problem. Compared with ensemble-averaged measurements, single-molecule force spectroscopy (SMFS) has evolved as a powerful technique to probe mechanical unfolding/folding of proteins at the single-molecule level, which provides rich details of protein conformational changes. Optical tweezers (OT), as one representative SMFS technique, have been widely used to directly monitor a protein folding process. This thesis presents a series of studies that use OT to investigate the mechanical unfolding/folding of two distinct classes of proteins: knotted/slipknotted proteins and Ca2+-binding metalloproteins. First, we combined OT and steered molecular dynamics (SMD) simulations to investigate a complex slipknotted protein. When stretched from the N- and C-termini, the slipknotted structure could be completely untied via multiple unfolding pathways. Upon relaxation, this protein showed complex folding behaviors involving misfolding. Our studies demonstrate the unfolding/folding mechanisms of a slipknotted protein with a complex topological conformation. Second, we used OT to mechanically tighten, untie and retie a trefoil knotted protein. Our results revealed that protein folding with a preformed knot was fast and robust; however, the folding from the unfolded polypeptide chain without the knot conformation was significantly slower, suggesting that the knotting was the rate-limiting step for the folding of this trefoil knotted protein. Next, we investigated the mechanical unfolding/folding behaviors of a Ca2+-binding protein, RTX (repeats-in-toxin) block V. Our results elucidated the secretion mechanism of this iv RTX block V and revealed that the mechanical design can ensure an efficient translocation process. Finally, we used OT to study another Ca2+-binding protein, RTX block IV. The mechanical properties of RTX block IV was well-characterized. Our results revealed that RTX block IV follows a similar translocation mechanism to be secreted as RTX block V. We also suggested that the secreted RTX block V is stabilized by folded RTX block IV and protected from being unfolded in vivo. Overall, this thesis clarified the unfolding/folding mechanisms of two knotted/slipknotted proteins and Ca2+-binding protein RTX. v Lay Summary Many biological processes involve unfolding and folding of proteins. Understanding protein unfolding and folding mechanisms has been one of the most important tasks in the field of the life sciences. As a powerful tool to probe the protein unfolding/folding dynamics at the single-molecule level, single-molecule force spectroscopy (SMFS) has been widely used to provide invaluable insights into the protein folding problem. By using a representative SMFS technique, optical tweezers (OT), we investigated the unfolding and folding mechanisms of two particular classes of proteins: knotted/slipknotted proteins and Ca2+-binding proteins. Our studies characterized the mechanical properties of these proteins, and provided a detailed picture of unfolding and folding mechanisms. Our studies also confirmed that structural features are highly related to protein function, setting the stage for development of new therapeutics for protein toxin-relevant diseases. vi Preface A version of chapter 2 has been published. Wang H., Gao X., Hu X., Hu X., Hu C. and Li H. (2019) Mechanical Unfolding and Folding of a Complex Slipknot Protein Probed by Using Optical Tweezers. Biochemistry, 2019, 58 (47), 4751-4760. This chapter is incorporated in this thesis with permission of the American Chemical Society, Copyright (2019). I designed the project together with Prof. Li. I conducted all the experiments and analyzed all the data from the OT experiments. I conducted all the SMD simulations and analyzed the data. Xiaoqing Gao, Xiaodong Hu, Xiaotang Hu and Chunguang Hu provided insightful feedback on experimental design and data analysis. I wrote the manuscript together with Prof. Li. A version of chapter 3 has been published. Wang H. and Li H. (2020) Mechanically Tightening, Untying and Retying a Protein Trefoil Knot by Single-Molecule Force Spectroscopy. Chem. Sci., 2020, 11(46), 12512-12521. This chapter is incorporated in this thesis with permission of the Royal Society of Chemistry, Copyright (2020). I designed the project together with Prof. Li. I conducted all the experiments and analyzed all the data from the OT experiments. I wrote the manuscript together with Prof. Li. A version of chapter 4 has been published. Wang H., Gao X., and Li H. (2019) Single Molecule Force Spectroscopy Reveals the Mechanical Design Governing the Efficient Translocation of the Bacterial Toxin Protein RTX. J. Am. Chem. Soc., 2019, 141 (51), 20498-20506. This chapter is incorporated in this thesis with permission of the American Chemical Society, Copyright (2019). I designed the project together with Prof. Li. I conducted all the experiments. Xiaoqing Gao developed programs by Igor Pro for data analysis. I and Xiaoqing Gao analyzed the data from the OT experiments. I wrote the manuscript together with Prof. Li. vii A version of chapter 5 is in preparation for publication. Wang H. and Li H. Single-Molecule Force Spectroscopy Reveals that the Mechanical Stability of Translocated RTX Domain Is Further Enhanced as the Secretion Proceeds. I designed the project together with Prof. Li. I conducted all the experiments and analyzed all the data from the OT experiments. I wrote this chapter. I wrote chapter 1 and 6. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables .............................................................................................................................. xiv List of Figures ...............................................................................................................................xv List of Symbols ........................................................................................................................... xix List of Abbreviations ................................................................................................................. xxi Acknowledgements .................................................................................................................. xxiv Dedication ................................................................................................................................. xxvi Chapter 1: Introduction ........................................................................................................... 1 1.1 Protein Structure and Protein Folding......................................................................... 3 1.1.1 Protein Structure ..................................................................................................... 3 1.1.2 Protein Folding........................................................................................................ 4 1.1.2.1 The Protein Folding Problem .......................................................................... 4 1.1.2.2 Protein Folding Models................................................................................... 5 1.1.2.3 Protein Folding Funnel ................................................................................... 7 1.1.2.4 Ensemble Studies on Protein Folding ............................................................. 9 1.1.2.5 Mechanical Unfolding and Folding of Proteins ............................................ 10 1.1.2.5.1 Knotted/slipknotted Proteins ................................................................... 11 1.1.2.5.2 Metalloproteins........................................................................................ 13 ix 1.2 Single-Molecule Force Spectroscopy (SMFS) ......................................................... 15 1.2.1 Principles of OT .................................................................................................... 15 1.2.2 SMFS Using OT ................................................................................................... 17 1.2.2.1 Constant-Force Mode.................................................................................... 19 1.2.2.2 Constant-Velocity Mode ............................................................................... 21 1.2.3 The Strengths and Limitations of OT ................................................................... 24 1.2.4 Review of Achievements on Protein Unfolding and Folding by OT.................... 25 1.2.4.1 Directly Monitoring Mechanical Folding of Proteins by OT ....................... 25 1.2.4.2 Probing Misfolding and Chaperone-Assisted Folding of Proteins ............... 27 1.2.4.3 Combining Fluorescence Spectroscopy and OT ........................................... 29 1.3 The Aim of This Thesis ............................................................................................ 31 Chapter 2: Mechanical Unfolding and Folding of a Complex Slipknotted Protein Probed by Using Optical Tweezers ..................................................................................................... 34 2.1 Synopsis .................................................................................................................... 34 2.2 Introduction ............................................................................................................... 35 2.3 Results and Discussion ............................................................................................. 39 2.3.1 Stretching PADC Leads to the Untying of the Slipknot and Complete Unfolding of PADC............................................................................................................................ 39 2.3.2 The Mechanical Unfolding of PADC Follows Multiple Pathways. ..................... 42 2.3.3 SMD Simulations of Mechanical Unfolding of PADC. ....................................... 43 2.3.4 Mechanical Unfolding Mechanism of PADC. ...................................................... 46 2.3.5 Untied and Unfolded PADC Can Fold Back into Its Native Slipknot Conformation: Complexity in the Folding of PADC. ....................................................... 48 x 2.4 Conclusions ............................................................................................................... 52 2.5 Experimental Section ................................................................................................ 53 2.5.1 Protein Engineering. ............................................................................................. 53 2.5.2 Preparation of dsDNA Handles and dsDNA-Protein Chimera. ............................ 55 2.5.3 Single-Molecule OT Experiments. ....................................................................... 56 2.5.4 Measuring Force-Extension Relationship of Protein Unfolding .......................... 58 2.5.5 Calculating the Unfolding and Folding Kinetics. ................................................. 59 2.5.6 SMD Simulations. ................................................................................................. 59 Chapter 3: Mechanically Tightening, Untying and Retying a Protein Trefoil Knot by Single-Molecule Force Spectroscopy ..................................................................................... 61 3.1 Synopsis .................................................................................................................... 61 3.2 Introduction ............................................................................................................... 61 3.3 Results ....................................................................................................................... 66 3.3.1 Stretching TrmD Leads to the Mechanical Unfolding of TrmD and the Tightening of the Trefoil Knot. ........................................................................................................... 66 3.3.2 The Mechanical Unfolding of TrmD Involved a Short-lived Intermediate State. 70 3.3.3 The Compaction of the Trefoil Knot. ................................................................... 71 3.3.4 The Unfolded and Knotted TrmD Can Refold Back to Its Native State Upon Relaxation. ........................................................................................................................ 73 3.3.5 Untying and Tying the Knotted Structure of TrmD Using OT ............................. 76 3.4 Discussion ................................................................................................................. 83 3.5 Conclusions ............................................................................................................... 86 3.6 Experimental Section ................................................................................................ 86 xi 3.6.1 Protein Engineering. ............................................................................................. 86 3.6.2 Preparation of DNA Handles and DNA-protein Chimera. ................................... 88 3.6.3 Single-Molecule OT Experiments. ....................................................................... 88 3.6.4 Measuring Force-Extension Relationship of Protein Unfolding .......................... 88 3.6.5 Calculating the Unfolding and Folding Kinetics. ................................................. 88 Chapter 4: Single-Molecule Force Spectroscopy Reveals the Mechanical Design Governing the Efficient Translocation of RTX Block V ..................................................... 90 4.1 Synopsis .................................................................................................................... 90 4.2 Introduction ............................................................................................................... 90 4.3 Results ....................................................................................................................... 94 4.3.1 Apo-RTX Block V Behaves as a Random Coil and Is Mechanically Compliant. 94 4.3.2 Holo-RTX block V Is Folded and Mechanically Stable. ...................................... 96 4.3.3 Unfolded RTX block V Can Refold Rapidly Against a Stretching Force Following Parallel Pathways........................................................................................... 100 4.3.4 Structure of the Unfolding and Folding Intermediate States. ............................. 101 4.3.5 Kinetics Analysis of Unfolding and Folding of RTX block V. .......................... 105 4.3.6 C-terminal α helix Stabilizes the Folded β-roll Structure and Facilitates Protein Folding. ........................................................................................................................... 106 4.3.7 Cation-π-π Interactions Play Important Roles in the Folding of C-terminal Structure. ......................................................................................................................... 107 4.4 Discussion ............................................................................................................... 109 4.4.1 Domain Structure of RTX block V Does Not Correspond to the Intermediate State Formed in the Unfolding/Folding Process. ..................................................................... 110 xii 4.4.2 Mechanical Design of RTX block V Ensures the Efficient Translocation. ........ 111 4.4.3 Translocation Mechanism of RTX block V. ....................................................... 113 4.5 Conclusions ............................................................................................................. 115 4.6 Experimental Section .............................................................................................. 116 4.6.1 Protein Engineering. ........................................................................................... 116 4.6.2 Preparation of DNA-protein Chimera. ................................................................ 117 4.6.3 Single-Molecule OT Experiment. ....................................................................... 117 4.6.4 Measuring Force-Extension Relationship of Protein Unfolding ........................ 117 4.6.5 Calculating the Unfolding and Folding Kinetics. ............................................... 118 Chapter 5: Single-Molecule Force Spectroscopy Reveals that the Mechanical Stability of Translocated RTX Domain Is Further Enhanced as the Secretion Proceeds ................. 119 5.1 Synopsis .................................................................................................................. 119 5.2 Introduction ............................................................................................................. 119 5.3 Results ..................................................................................................................... 123 5.3.1 RTX Block IV Does Not Fold Independently. ................................................... 123 5.3.2 Folded RTX Block V Is a Crucial Prerequisite for Folding of RTX Block IV. . 125 5.3.3 The Mechanical Unfolding of RTX Block IV Involves the Formation of Multiple Intermediate States. ......................................................................................................... 132 5.3.4 Unfolded RTX Block IV Can Fold Back to the Native State Following Parallel Pathways. ........................................................................................................................ 134 5.3.5 The Unfolding/Folding Mechanism of RTX Block IV. ..................................... 135 5.3.6 RTX Block IV-V Displays a Different Mechanical Stability Between Its N- and C-terminus....................................................................................................................... 140 xiii 5.3.7 RTX Block IV and V Can Interact and Stabilize Each Other. ............................ 149 5.4 Discussion ............................................................................................................... 153 5.4.1 Folded RTX Block V Is Necessary for the Folding of RTX Block IV............... 153 5.4.2 Folded RTX Block IV in Turn Stabilizes the Translocated RTX Block V. ....... 153 5.5 Conclusions ............................................................................................................. 154 5.6 Experimental Section .............................................................................................. 155 5.6.1 Protein Engineering. ........................................................................................... 155 5.6.2 Preparation of DNA-protein Chimera. ................................................................ 156 5.6.3 Single-Molecule OT Experiment. ....................................................................... 156 5.6.4 Measuring Force-Extension Relationship of Protein Unfolding ........................ 156 5.6.5 Calculating the Unfolding and Folding Kinetics. ............................................... 156 Chapter 6: Summary and Future Work ............................................................................. 157 6.1 Thesis Summary...................................................................................................... 157 6.2 Future Work and Outlook ....................................................................................... 159 Bibliography ...............................................................................................................................163 Appendix .....................................................................................................................................178 xiv List of Tables Table 3.1 Average unfolding and folding forces and kinetic parameters for TrmD9,246 and TrmD Δhelix45,128 ..................................................................................................................................... 68 Table 4.1 Average unfolding and folding forces and kinetic parameters for wt RTX block V and RTX Δhelix1529,1664 ...................................................................................................................... 100 Table 5.1 Percentage of each pathway via which the unfolding/folding of RTX block IV occurs..................................................................................................................................................... 132 Table 5.2 Average unfolding and folding forces and kinetic parameters for RTX block IV ..... 133 xv List of Figures Figure 1.1 The four levels of structural hierarchy in a protein. ..................................................... 3 Figure 1.2 Models of protein folding: diffusion-collision model (left), nucleation-condensation model (middle) and hydrophobic-collapse model (right). .............................................................. 6 Figure 1.3 A protein folding funnel with a rugged energy surface. ............................................... 7 Figure 1.4 The effect of mechanical force on the energy landscape for a two-state system. ........ 8 Figure 1.5 3D structures of some representative protein knots.................................................... 12 Figure 1.6 3D structures of some representative Ca2+-binding proteins. ..................................... 15 Figure 1.7 Schematics showing the principles of OT. ................................................................. 17 Figure 1.8 Schematics of the OT experiment to study a polyprotein GB1 (PDB: 2QMT) tetramer. ........................................................................................................................................ 18 Figure 1.9 A representative extension-time curve of a prion protein in constant-force mode. ... 19 Figure 1.10 Unfolding probability density for all unfolding time intervals with a single exponential fit (black line; dotted lines represent 95% confidence intervals of the fit). ............... 21 Figure 1.11 A representative force-extension curve showing the unfolding/folding of a prion protein. .......................................................................................................................................... 22 Figure 1.12 Direct observation of protein folding in the OT experiments. ................................. 26 Figure 1.13 Protein EF-G and experimental schematics of OT to stretch the ribosome-nascent chain complex. .............................................................................................................................. 29 Figure 1.14 Combining fluorescence spectroscopy and OT to study protein unfolding and folding. .......................................................................................................................................... 31 Figure 2.1 Stretching and relaxing a slipknotted protein by using OT. ....................................... 37 xvi Figure 2.2 Stretching PADC from its N- and C-termini leads to the untying of the slipknot and the complete unfolding of PADC. ................................................................................................ 41 Figure 2.3 Three-state unfolding of PADC. ................................................................................. 43 Figure 2.4 SMD simulations of the mechanical unfolding of PADC. ......................................... 45 Figure 2.5 Unfolded PADC can fold back to its native slipknotted state. ................................... 49 Figure 2.6 Coomassie blue stained SDS-PAGE gel of the purified NuG2-PADC-NuG2. .......... 55 Figure 2.7 Schematics of the optical setup used in the MiniTweezers. ....................................... 57 Figure 3.1 Stretching and relaxing TrmD using OT. ................................................................... 64 Figure 3.2 Stretching TrmD9,246 to a tightened trefoil knot involves two parallel pathways. ...... 67 Figure 3.3 Mechanical unfolding and folding of TrmD Δhelix9,161. ............................................ 69 Figure 3.4 Representative force-distance curves showed the mechanical unfolding of TrmD9,246 at low forces. ................................................................................................................................. 72 Figure 3.5 Unfolded and tightened TrmD9,246 can fold back to its native state. .......................... 74 Figure 3.6 Mechanical unfolding and folding of two NuG2 protein domains at a pulling speed of 50 nm/s. ......................................................................................................................................... 75 Figure 3.7 Untying and tying the knotting loop of TrmD Δhelix45,128. ........................................ 77 Figure 3.8 Force-extension relationship of the unfolding of TrmD Δhelix45,128. ......................... 78 Figure 3.9 Stretching the protein TrmD45,128 does not untie the knotted conformation. .............. 81 Figure 3.10 High tension does not inhibit the folding of TrmD9,246. ........................................... 85 Figure 3.11 Coomassie blue stained SDS-PAGE gel of different constructs of TrmD. .............. 88 Figure 4.1 Probing the unfolding and folding of RTX block V using OT. .................................. 93 Figure 4.2 Far-UV CD spectra shows that RTX block V is unstructured in the absence of Ca2+ (solid curve) and is folded in the presence 10 mM Ca2+ (dashed curve). ..................................... 94 xvii Figure 4.3 Representative force-distance curves showing the unfolding/folding of Nug2-apo-RTX block V-NuG2 at a pulling speed of 50 nm/s. ..................................................................... 96 Figure 4.4 Holo-RTX block V is mechanically stable and unfolds in two parallel pathways. .... 97 Figure 4.5 OT experiments showed that the deletion of α helix at the C-terminus does not affect the unfolding and refolding pathways of RTX block V. .............................................................. 99 Figure 4.6 OT experiments revealed the structure of unfolding/folding intermediate state. ..... 102 Figure 4.7 Schematics of two-state and three-state unfolding/folding pathways of RTX block V...................................................................................................................................................... 104 Figure 4.8 Force-dependency of the unfolding and refolding rate constants of RTX block V for the pathways of (A) N ↔U, (B) N ↔I and (C) I ↔U. ............................................................... 105 Figure 4.9 Signatures of the mechanical unfolding (red) and refolding (blue) of RTX Δhelix1529,1664. .............................................................................................................................. 107 Figure 4.10 Mechanical unfolding and folding of RTX block V W1645G-Y1646G are investigated by OT. ..................................................................................................................... 108 Figure 4.11 Cation-π-π interaction is critical for the rapid folding of RTX block V................. 109 Figure 4.12 Schematics of proposed translocation mechanism of RTX block V. ..................... 114 Figure 4.13 Coomassie blue stained SDS-PAGE gel of different constructs of RTX block V. 117 Figure 5.1 Probing the unfolding and folding of RTX block IV using OT. .............................. 122 Figure 5.2 Representative force-distance curves showing the unfolding/folding of NuG2-apo-RTX block IV-NuG2 (left) and NuG2-apo-RTX block IV-V-NuG2 (right) at a pulling speed of 50 nm/s. ....................................................................................................................................... 124 Figure 5.3 Representative curves showing the unfolding/folding of NuG-RTX block IV-NuG2 at the pulling speed of 50 nm/s. ...................................................................................................... 125 xviii Figure 5.4 Holo-RTX block IV-V is mechanically stable and can fold rapidly. ....................... 127 Figure 5.5 Zoomed-in view of the second unfolding event of RTX block IV-V. ..................... 128 Figure 5.6 Mechanical unfolding/folding of RTX block IV1396,1528-V. ..................................... 130 Figure 5.7 Mechanical unfolding/folding of RTX block IV1411,1528-V. ..................................... 131 Figure 5.8 Force-dependency of the unfolding (red) and folding (blue) rate constants of RTX block IV for the pathways of (A) N↔I1, (B) I1↔I2 and (C) I2↔U. ........................................ 134 Figure 5.9 Unfolding/folding of RTX block IV1429,1528 occurs via a three-state pathway. ........ 137 Figure 5.10 Schematics showing the unfolding and folding pathways of RTX block IV. ........ 139 Figure 5.11 Mechanical unfolding/folding of RTX block IV1429,1528-V. ................................... 142 Figure 5.12 Mechanical unfolding/folding of RTX block IV1447,1528-V. ................................... 144 Figure 5.13 Mechanical unfolding/folding of RTX block IV1465,1528-V. ................................... 145 Figure 5.14 Mechanical unfolding/folding of RTX block IV1483,1528-V. ................................... 146 Figure 5.15 RTX block IV-V displays a different mechanical stability between its N- and C-terminus....................................................................................................................................... 148 Figure 5.16 Force-dependency of the unfolding rate constants of the unfolding of RTX block V when RTX block IV is folded. .................................................................................................... 151 Figure 5.17 Force-dependency of the unfolding rate constants of the unfolding of RTX block IV1447,1528 when RTX block V is unfolded (blue) and folded (red). ........................................... 152 Figure 6.1 Schematics of a tightened slipknot configuration of thymidine kinase. ................... 160 xix List of Symbols α0 Unfolding rate at zero force α(F) Unfolding rate at the force of F β0 Folding rate at zero force β(F) Folding rate at the force of F dPu(t)/dt Unfolding probability density dPf(t)/dt Folding probability density ΔG Change in Gibbs free energy Δxu Unfolding distance, the distance between native and transition state Δxf Folding distance, the distance between unfolded and transition state ΔLc Contour length increment of protein unfolding F (x) Entropic force at the extension of x h Plank constant κ Transmission coefficient k(f) Rate constant at a given force f kB Boltzmann constant Lc Contour length N(f) Total number of observed unfolding/folding events under a given force f p Persistence length Pu(t) Unfolding probability Pf(t) Folding probability t Time xx t(f) Total dwell time under a given force f T Temperature in Kelvin x Protein extension xxi List of Abbreviations aa Amino acid AdoMet S-adenosyl-L-methionine AFM Atomic force microscopy AFV3-109 The ORF109 of the Acidianus Filamentous Virus 3 ATP Adenosine triphosphate bp Base pair CaM Calmodulin CD Circular dichroism CyaA Adenylate cyclase DNA Deoxyribonucleic acid dsDNA Double-strand deoxyribonucleic acid E Extension E. coli Escherichia coli EF-G Elongation factor G F Force FRET Fluorescence resonance energy transfer GB1 The B1 binding domain of protein G from streptococcus GFP Green fluorescent protein HCl Hydrochloric acid I Intermediate state of protein IPTG Isopropyl β-D-1-thiogalactopyranoside xxii LB Luria-Bertani broth N Native state of protein MD Molecular dynamics mM Millimolar MT Magnetic tweezers MTases Methyltransferases nm Nanometer NMR Nuclear magnetic resonance NuG2 A designed variant of protein G OD Optical density OT Optical tweezers PADC Pyruvoyl-dependent arginine decarboxylase PBS Phosphate buffered saline PDB Protein data bank PCR Polymerase chain reaction pN Piconewton RNA Ribonucleic acid RTX Repeats-in-toxin SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SMD Steered molecular dynamics SMFS Single-molecule force spectroscopy T1SS Type I secretion system TCEP Tris(2-carboxyethyl)phosphine xxiii Tris Tris(hydroxymethyl)aminomethane TrmD Escherichia coli tRNA (guanosine-1) methyltransferase D tRNA Transfer ribonucleic acid TF Trigger factor U Unfolded state of protein UCH-L1 Ubiquitin C-terminal hydrolase isoenzyme L1 VMD Visual Molecular Dynamics wt wild-type WLC Worm-like chain 3D Three-dimensional xxiv Acknowledgements I would like to express my earnest appreciation to my supervisor, Dr. Hongbin, Li, for his guidance and support throughout my journey to pursue a Ph. D. degree. I would not be who I am right now without his continuous encouragement and selfless assistance. His knowledge, expertise and dedication to work not only made him a real scientist, but also inspired me to work hard and make progress. This experience will definitely be invaluable for my life and I am eternally grateful for everything he has taught me. I would like to thank all the members in Li’s group. Thank you to Dr. Chengzhi He for teaching me the molecular biology, the steered molecular dynamics simulations and the optical tweezers technique. Thank you to Dr. Xiaoye Gao, Dr. Na Kong, Kailin Zhou, Jinliang Li, Dr. Hai Lei, Dr. Ao Xia, Dr. Shanshan Lyu, Yabin Guo, Bryce Laver, Xiaoqing Gao, Dr. Ruidi Wang, Dr. Yongliang Wang, Dr. Jiahao Xia, Dr. Wenxu Sun, Linglan Fu, Jiayu Li, Tianyu Duan, Adam Xiao, Haoquan Lin, Jiacheng Zuo, Qingyuan Bian, Jess Fong and Guojun Chen for their help and friendship. I would like to appreciate the help from my collaborators during my internship. Many thanks to Dr. Chunguang Hu, Dr. Wanfu Shen, Dr. Shuai Li, Guoteng Ma and Xiao Su for the kind help in Tianjin University. Thank you to Dr. Peng Chen, Dr. Xiao Xie, William Ngai, Huangtao Zheng, Jiaofeng Li and Yuange Duan for their unselfish assistance and support in Peking University. I would like express my appreciation to Dr. Suzana K Straus, Dr. Katherine S. Ryan and Dr. Derek P Gates for their valuable suggestions and intellectual guidance as members of my supervisory committee. I would also like to thank Dr. Suzana K Straus, Dr. Katherine S. Ryan xxv for critically reading my thesis and providing valuable feedbacks for improving the readability of this thesis. Many thanks to Zhengshun Qiao and Guodong Wang, who have provided me a lot of help during my time in Vancouver. A special thanks to my landlord, Wei Chen, for the numerous times she helped and supported me for the past six years. I feel really fortunate to live here for such a long time. Thanks to all my friends, may the joy and happiness be around you today and always. Last but not least, I want to thank my parents for their deep love and endless support. xxvi Dedication To My Parents1 Chapter 1: Introduction Proteins are complex biomacromolecules that perform essential physiological functions in nearly every biological process in living organisms, including cell signaling, molecular transport, catalysis of biological reactions and immunity to foreign substances.1-3 These various protein functions originate from the structural diversity of proteins. Currently, over 150,000 protein structures have been solved, and that number is continually rising.4 All proteins are made of one building block: the amino acid. Twenty different amino acids can be combined to form almost every type of protein in living organisms. Therefore, understanding the molecular mechanism of how proteins can fold into distinct structures has been one of the most important questions in the field of biochemistry and remains a significant challenge. Protein folding starts from a sequence of amino acids, also known as a polypeptide, to a well-defined three-dimensional (3D) structure. Under physiological conditions, most proteins are able to fold spontaneously into their correct structures within a short time after being synthesized and remain stably folded. Since the 1960s, with the appearance of the first atomic-resolution protein structure of myoglobin,5-6 the notion of a “folding problem” has emerged and has attracted tremendous interest. The protein folding problem boils down to three main questions: (i) How does a protein’s amino acid sequence dictate the protein’s 3D structure? (ii) How does a protein fold into its native structure so quickly? (iii) Can we predict a protein structure from its original sequence using a computer algorithm?7-9 The protein folding problem was viewed as a great challenge in the field of life sciences. Solving this problem required enormous advances in experimental and theoretical methods. Traditional studies were primarily based on ensemble methods.10-14 However, the knowledge obtained from ensemble studies was based on averaged properties of a large quantity of protein 2 molecules, and the molecular details associated with a single protein were obliterated. Over the past two decades, single-molecule force spectroscopy (SMFS) techniques have rapidly evolved into powerful tools for investigating protein unfolding/folding dynamics at the single-molecule level. By measuring the inter/intramolecular interactions, mechanical unfolding/folding of a single protein can be directly investigated. SMFS studies have provided invaluable insights into the folding of proteins that are almost inaccessible via traditional ensemble measurements.15-22 SMFS can be performed using several different techniques, including atomic force microscopy (AFM), optical tweezers (OT) and magnetic tweezers (MT). In this thesis, I will only focus on the mechanical unfolding/folding of single proteins by OT. In this chapter, I will provide a review of this newly developed research area. It will contain the following sections: 1). Introduction of the basic knowledge relating to protein structure and several widely accepted protein folding theories. In this part, I will discuss the fundamental concepts of the protein and the achieved progress made in the comprehension of protein folding processes. 2). SMFS via OT and its application to protein mechanical unfolding/folding studies. In this section, I will introduce the techniques of SMFS, particularly OT. I will explain details relating to the basic principle of OT and how OT is applied in the SMFS experiment. To show the advantages of OT in protein folding studies, I will also summarize some representative achievements that OT has accomplished in the mechanical unfolding/folding of proteins. 3). The aim of this thesis will be introduced. 3 1.1 Protein Structure and Protein Folding 1.1.1 Protein Structure Most proteins are a linear polypeptide chain built from L-amino acids, of which there are twenty different types. Four levels of structural hierarchy are often used to depict protein structure, i.e., the primary structure to the quaternary structure (Figure 1.1). The primary structure is the linear sequence of amino acids within a polypeptide chain. It is the arrangement of the amino acid types, i.e., which amino acid follows the next in the sequence of a given protein.23-24 The secondary structure refers to local folded structural subunits, stabilized by hydrogen bonds between adjacent amino acid residues in the polypeptide backbone. The most representative types of secondary structures are the α helix and the β sheet.25 The tertiary structure is a combination of multiple secondary structures within a single polypeptide chain.26 The quaternary structure refers to the association of multiple polypeptide chains that have tertiary structures in a particular manner.27 Figure 1.1 The four levels of structural hierarchy in a protein. The 3D structures are generated and shown using Visual Molecular Dynamics (VMD).28 The tertiary and quaternary structures are adapted from the human hemoglobin protein (PDB 1A3N). The protein structure is usually stable under physiological conditions. A series of interactions within the protein play critical roles in maintaining its folded structure, including 4 electrostatic, van der Waals, hydrophobic, hydrogen bonding interactions and disulfide bonds.29 The stability of a protein provides the basis for structural diversity. X-ray crystallography30, nuclear magnetic resonance31 and single-particle cryogenic electron microscopy32 are used to solve the protein structures. The structural information is subsequently deposited into a database called the Protein Data Bank (PDB).4 The overall number of protein structures released in the PDB is increasing annually.33 1.1.2 Protein Folding 1.1.2.1 The Protein Folding Problem Understanding the manner in which proteins fold has been one of the most important tasks in the field of biochemistry. Extensive efforts have been made to address the protein folding problem.34-41 A major milestone in solving this challenge was the thermodynamic hypothesis (also known as Anfinsen’s dogma) proposed by Christian Anfinsen and his colleagues.23-24 From the well-known experiments on the folding of ribonuclease A, they concluded that the native state of protein was thermodynamically stable under physiological conditions and that natural conformation was only determined by the amino acid sequence (i.e., primary structure). Their studies shed light on the relationship between primary structure and tertiary structure and led to the development of protein folding theories. However, the thermodynamic hypothesis only illustrated whether protein folding is feasible, but did not address the question of how a protein is able to access its native state within a biologically relevant timescale. In practice, a protein might sample a large number of possible conformations until it reaches the correct conformation. One challenge, proposed by Cyrus Levinthal (also known as Levinthal’s paradox),39 is that it would take a time longer than the age of the universe for a protein to find the correct folded structure during its folding process, assuming the protein sampled this conformational space randomly. The 5 result of this thought experiment was evidently contrary to the actual protein folding time, which is from milliseconds to a few hours (hence the paradox). Therefore, protein folding must occur via a certain mechanism that avoids random sampling and that must be confined to a limited number of conformations. 1.1.2.2 Protein Folding Models Inspired by Levinthal’s paradox, several protein folding models have since been proposed to describe how proteins might fold via a specific route rather than by simple random sampling of every possible conformation. Three representative protein folding models are: 1) the diffusion-collision model;37-38 2) the nucleation-condensation model36; and 3) the hydrophobic-collapse model42 (Figure 1.2). In the diffusion-collision model, the first step in the folding process involves the formation of secondary structural elements. These secondary structures then move diffusely and collide with each other to coalesce into a final tertiary structure. The nucleation-condensation model, on the other hand, assumes that protein folding initiates from the formation of a metastable nucleus, around which the surrounding polypeptide is then rapidly condensed and folded into the final structure. The nucleation-condensation model differs from the diffusion-collision model in that the secondary and tertiary structures are formed concurrently.43 Finally, the hydrophobic-collapse model assumes that the hydrophobic force is the primary driving force for the folding of a globular protein. In this model, the polypeptide chain rapidly collapses into a “molten globule” state, followed by the formation of secondary structure.44 6 Figure 1.2 Models of protein folding: diffusion-collision model (left), nucleation-condensation model (middle) and hydrophobic-collapse model (right). Image adapted with permission from Elsevier, Copyright © 2002.44 These protein folding models incorporate driving forces, which help to rationalize why proteins fold on timescales that are much shorter than those associated with a random sampling of conformations, i.e., Levinthal’s paradox. In addition to the three models mentioned above, other protein folding models have also been proposed, such as the framework model.45 It should be noted that these models are not mutually exclusive and that some models share many similarities.43 These protein folding models have been supported by experimental evidence and have provided a straightforward picture of how proteins fold.40-41 However, these models of protein folding are mostly oversimplified and solely highlight one or a few aspects of the folding process for simple proteins. Therefore, these models are not sufficient to describe the folding of a large proportion of proteins that have more complicated structures. To better characterize a general protein folding mechanism, a complete quantitative description of the folding process is required, including conformational changes, energetics and 7 kinetics. A new view of protein folding was developed by Wolynes and others,46 making use of an energy landscape and a protein folding funnel. This protein folding model has evolved over the past twenty years as a result of both experimental and computational studies and has progressively replaced the classical models of a sequential and hierarchical folding pathway.47 1.1.2.3 Protein Folding Funnel An energy landscape has been an effective tool to describe chemical reaction dynamics.48-50 Protein folding is a process involving the rearrangement of structural elements and a minimization of total free energy, making it suitable for energy landscape theory.34-35 Previous studies have suggested that the energy landscape for protein folding is funnel-shaped, i.e., a protein folding funnel (Figure 1.3).51-52 Figure 1.3 A protein folding funnel with a rugged energy surface. Image adapted with permission from Springer Nature, Copyright © 1997.52 The depth of the funnel represents the free energy of a given conformational state; the width of the funnel measures the configurational entropy. In the protein folding funnel model, protein folding can be described as a process that goes from high-energy conformations (i.e., highly disordered states) toward a conformation that is at the bottom of the funnel (i.e., the native state) via multiple routes. An ideal funnel has a smooth energy landscape, in which protein folding occurs quickly. However, real funnels are often rugged. The rugged energy landscape consists of 8 many nonnative local minima, separated by energy barriers with different heights. These nonnative local minima are also known as kinetic traps that often result in slow folding.53 The presence of nonnative local minima in the funnel suggests that either on-pathway intermediate states or off-pathway intermediate states (i.e., misfolded states) exist during the folding process.54 In particular, many small proteins have been found to fold in a two-state manner, and only two conformational states (i.e., the native state and the unfolded state) populate the experiments. The transition between the two states involves overcoming only one energy barrier. The saddle point of the free energy profile is defined as the transition state (Figure 1.4, black curve). Figure 1.4 The effect of mechanical force on the energy landscape for a two-state system. At zero force, the native state has a lower energy and is more stable (black curve). In the presence of an external force, the energy landscape is tilted until the unfolded state is more stable (blue curve). The unfolding and folding distances are indicated by Δxu and Δxf, respectively. The transition state is indicated by ǂ. Image adapted with permission from John Wiley and Sons, Copyright © 2013.55 The protein folding funnel theory has now been widely accepted to describe protein folding and has also been used to describe other biological processes, such as protein-ligand binding.56-58 9 1.1.2.4 Ensemble Studies on Protein Folding Extensive experimental efforts have been expended to investigate protein folding. In ensemble studies, fluorescence or circular dichroism (CD) signals were measured to monitor conformational changes in proteins.59 In ensemble studies, protein folding can be initiated from an unstructured polypeptide. To obtain the unfolded protein, the native protein was unfolded using denaturants, such as guanidine hydrochloride,60-61 urea,62-63 heat64-65 and extreme pH66. Denaturants alter the energy landscape until the energy of the unfolded state is lower than that of the native state, leading to spontaneous protein unfolding. Notably, protein unfolding might occur through different routes under different denaturing conditions66. After restoring normal physiological conditions, e.g., removing chemical denaturants from the system60-63 or adjusting the temperature back to room temperature64, protein folding can be initiated. Previous studies based on ensemble methods have provided considerable insights into protein folding mechanisms, as well as folding thermodynamics and kinetics.10-14 Since protein folding was probed in ensemble-averaged measurements, rare or transient conformational changes associated with a single protein are difficult to detect. Compared to ensemble methods, single-molecule studies opened up the possibility of exploring the molecular details of protein folding. Over the past twenty years, SMFS has evolved as a powerful tool to study protein unfolding and folding from a mechanical perspective at the single molecule level. In SMFS studies, force-induced protein unfolding (or folding) can be triggered by stretching (or relaxing) a single protein using SMFS techniques, such as OT and AFM. SMFS studies make use of mechanical force as a denaturant to investigate protein unfolding/folding at the single-molecule level, setting a new stage for solving the protein folding problem.67 10 1.1.2.5 Mechanical Unfolding and Folding of Proteins Force-induced biological processes are involved in many facets of cellular activities, including DNA replication68-69 and transcription,68, 70 RNA translation,68 protein folding,71 translocation,72 and degradation,73 in which mechanical force plays an important role. Mechanical force is considered not only as a product of a biological reaction but also as an inducer for triggering conformational changes in biomolecules. Therefore, characterizing the mechanical properties of proteins is important for elucidating the mechanism underlying many biological processes. Mechanical force can induce protein unfolding. Similar to chemical denaturants, mechanical force applied on proteins can lead to a tilted energy landscape, in which the unfolded state becomes more stable than the native state. Moreover, mechanical force can lower the energy barrier height, thereby speeding up the unfolding reaction (Figure 1.4, blue curve). The effect of mechanical force on the energy landscape is described by the Bell-Evans model.74-75 𝛼(𝐹) = 𝜅𝑘𝐵𝑇ℎ exp (−𝛥𝐺𝑇−𝑁−𝐹𝛥𝑥𝑢𝑘𝐵𝑇) = 𝛼0 exp (𝐹𝛥𝑥𝑢𝑘𝐵𝑇) (1.1) 𝛽(𝐹) = 𝜅𝑘𝐵𝑇ℎexp (−𝛥𝐺𝑇−𝑈+𝐹𝛥𝑥𝑓𝑘𝐵𝑇) = 𝛽0 exp (−𝐹𝛥𝑥𝑓𝑘𝐵𝑇) (1.2) where F is the mechanical force, and α(F) and β(F) are the unfolding and folding rate constants at the given force F, respectively. α0 and β0 are the intrinsic unfolding and folding rate constants at zero force, respectively. Δxu (unfolding distance) is the distance between the native state and the transition state, and Δxf (folding distance) is the distance between the unfolded state and the 11 transition state. kB is the Boltzmann constant, T is the absolute temperature, κ is the transmission coefficient and h is Plank’s constant. It is important to note that protein folding in vivo is quite different from that in vitro.76-78 In contrast to in vitro experiments, where protein folding begins from the full-length unfolded polypeptide, protein folding in vivo may take place on the ribosome when protein synthesis is still in progress.77 The ribosomes have been revealed to play important roles in protein folding by mediating the folding rates.78-79 Additionally, in many cases, protein folding in vivo requires the assistance of chaperones. These chaperones can interact with the nascent polypeptide chain and guide the folding to the correctly folded structure, as well as prevent protein misfolding.79 Another difference between protein folding in vivo and in vitro lies in the effects of macromolecular crowding.80-82 Proteins are synthesized in environments that are crowded with macromolecules and their assemblies, which can prevent the aggregation of unfolded polypeptides and misfolding of proteins.80 Many in vitro/computational studies have revealed that crowding usually results in a higher thermodynamic stability and faster folding kinetics, despite that the absolute effects of crowding agents are small.80-81 Understanding the unfolding/folding mechanism of proteins in vivo remains a challenge and requires further efforts. In this thesis, I will only focus on the protein unfolding and folding in vitro. In the following chapters, SMFS method was employed to investigate the mechanical properties of two particular classes of proteins: knotted/slipknotted proteins and metalloproteins. 1.1.2.5.1 Knotted/slipknotted Proteins The knotted conformation in the polypeptide backbone is one of the most surprising topological elements found in proteins in the last twenty years. In fact, given the complexity of the knotted geometry, the existence of protein knots was once a matter of debate.83-85 Since the late 12 1990s, proteins have been discovered to possess knotted or slipknotted conformations within their structure.83, 86-87 The first discovery of a knotted protein, human carbonic anhydrase, was made by Mansfield in 1994.83 Currently, over 1,300 proteins have been identified as knotted or slipknotted proteins in the PDB, including trefoil (31), figure-of-eight (41), Gordian (52) and Stevedore (61) knots (Figure 1.5).88-90 This complicated structural feature was suggested to play a significant role in maintaining protein stability, as well as allowing proteins to perform their functions effectively.88, 91-93 Understanding the molecular mechanism of how knotted proteins overcome topological barriers in the folding process has been a significant challenge and is yet to be fully elucidated. Figure 1.5 3D structures of some representative protein knots. From left to right: (A) YbeA, a methyltransferase from E. coli (PDB: 1NS5); (B) E. coli class II ketol-acid reductoisomerase (PDB: 1YRL); (C) Human ubiquitin carboxy-terminal hydrolase L1 (PDB: 2ETL); and (D) α haloacid dehalogenase (PDB: 4N2X). Here, the Alexander-Briggs notation (xy) is used to present different types of knots. The first number indicates the minimum crossing number of a given knot, and the second number written as subscript denotes the serial reference number. Image adapted with permission from IOP Publishing Ltd, Copyright © 2015.89 13 The formation of a protein knot requires the topological entanglement of a linear polypeptide. Taking the formation of a trefoil knot as an example: one terminal end of the polypeptide bends and forms a closed loop, while the other end is guided and threaded inside. Disulfide bonds and metal ions within the protein may also assist in the formation of the knotted structure.86 Stretching a folded protein with a knotted structure from the N- and C-termini can lead to a compact and tightened knot within the unfolded protein chain. The slipknotted conformations require incomplete back-threading of one terminal end through the knotted loop. Upon stretching from the N- and C-termini, instead of being extended into a tightened knot, the slipknotted conformation can be completely untied. Despite their structural differences, knotted and slipknotted structural elements have been suggested to be related, e.g., the slipknot can serve as an important intermediate state during the folding of knotted proteins, such as the knotted protein MJ0366.94 Chapters 2 and 3 of this thesis are focused on studies of two proteins with knotted/slipknotted conformations, including one slipknotted protein, pyruvoyl-dependent arginine decarboxylase (PADC) (PDB: 2QQD), and one knotted protein, Escherichia coli tRNA (guanosine-1) methyltransferase D (TrmD) (PDB: 1P9P). 1.1.2.5.2 Metalloproteins Metalloproteins are ubiquitous in nature and account for approximately half of all proteins found in nature.95-96 A wide range of metal elements are incorporated into proteins, including iron,97-98 copper,99-100 zinc,101 calcium,102-103 and manganese.104 Protein-ion interactions are primarily governed by electrostatic forces or coordination bonds,105 which can further enhance protein stability. The metal in the protein is responsible for many critical enzymatic activities, including photosynthesis, oxygen transport and nitrogen fixation,95 thereby playing an essential role in cellular life. 14 Calcium is necessary to maintain the life of cells and organisms. In Ca2+-binding proteins, Ca2+ ions are often coordinated by carbonyl or carboxy groups of polar amino acid residues, such as aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn) and glutamine (Gln). Noncovalent charge-dipole interactions maintain the protein-Ca2+ complex.106 Therefore, Ca2+-binding proteins are often less stable than other metalloproteins, in which the protein-metal complex is maintained by coordination bonds, e.g., thiol-metal bonds. A large number of Ca2+-binding proteins have been found to be important in regulating many cellular processes (Figure 1.6).107 For example, calmodulin (CaM) is crucial in many cell signaling pathways. CaM binds more than 300 target proteins in a Ca2+-dependent manner, such as kinases, phosphatases, ATPases and ion channels.108 Binding can induce conformational changes in target proteins, leading to a change in their response to Ca2+ concentration. In this way, CaM regulates the function of target proteins, thus playing a crucial role in Ca2+-mediated signal transduction.103 Other Ca2+-binding proteins include protein toxins, which are virulent to the life of cells.109-114 These protein toxins can create pores in cell membranes and quickly ablate the physiological functions of host cells.112-114 The bacterial toxin adenylate cyclase toxin (CyaA) is an example of such a protein toxin. CyaA is a large protein consisting of 1706 amino acid residues. CyaA bears five consecutive protein domains at the C-terminus, named RTX (repeats-in-toxin) blocks. Each RTX domain incorporates Ca2+ ions to fold into a β-roll structure. Chapters 4 and 5 of this thesis are focused on studies of two RTX domains, RTX block IV and V, revealing how the mechanical design of the protein toxin may facilitate its secretion. 15 Figure 1.6 3D structures of some representative Ca2+-binding proteins. From left to right: parvalbumin, calmodulin and RTX block V. 1.2 Single-Molecule Force Spectroscopy (SMFS) SMFS has paved the way to directly monitor the mechanical unfolding/folding process of proteins at the single-molecule level, and has provided invaluable details associated with protein unfolding/folding.67, 115-119 AFM, OT and MT are three representative tools that have been widely used.67, 115, 117 Each individual technique has its own technical features and can be exploited accordingly.67 In particular, the superb resolution in force (0.1 pN) makes OT an ideal technique for directly observing the protein folding process.120 In this thesis, I used OT to investigate the mechanical properties of proteins at the single-molecule level. 1.2.1 Principles of OT The wave-particle duality of light states that light not only exhibits wavelike properties but also harbors characteristics of particles. This hypothesis has been supported by many experimental efforts and has provided the basis for the development of OT.121 Under illumination, an object experiences radiation pressure exerted by photons (i.e., light particles) due to the momentum exchange between the photon and object. Radiation pressure was first suggested by Johannes Kepler in 1619 and was subsequently experimentally demonstrated in 1901.122 In 1970, the application of radiation pressure was first realized by Arthur Ashkin, who invented OT to trap and 16 manipulate tiny objects.123 Due to his seminal contributions to the development of OT and its applications in physics and biology, he was awarded the Nobel Prize in Physics in 2018. OT provides a noncontact approach to trap and manipulate microscopic objects, such as silica beads, plastic particles, single cells and even small living biological species. Figure 1.7 displays the basic principle of how OT exerts forces to hold an object in place.124 To form an optical trap, a high-intensity laser is used as the light source and is then tightly focused by an objective lens with a high numerical aperture. The manipulated object (e.g., polystyrene bead) should have a higher refractive index than that associated with the surrounding medium. The optical force exerted on a bead can be deconstructed into two components: a gradient force and a scattering force. The gradient force is generated from the refraction of photons passing through the bead. If the bead is laterally displaced from the focal spot, it will deflect the incident light away from the direction of the light propagation. This change in light direction is accompanied by a difference in light momentum flux, generating a restoring force (i.e., gradient force) to pull the bead towards the focus. Similarly, if the bead is displaced axially, the refracted rays will also generate a restoring force to pull or push the bead back. The scattering force results from the reflection of photons on the surface of the bead. The scattering force tends to push the bead in the direction of light propagation, slightly displacing the trapped bead downstream from the focused center. Therefore, the small bead is trapped in an equilibrium position slightly off the focal spot, where the gradient force and the scattering force are balanced. In some OT setups, two counterpropagating laser beams are used to overcome the scattering force to create more stable trapping.125 17 Figure 1.7 Schematics showing the principles of OT. (A) Refraction of photons induces a momentum exchange between the laser and the bead. (B) Ray optics model showing how a focused laser beam can tightly trap the bead in the focus. Image adapted with permission from De Gruyter, Copyright © 2019.124 OT makes it possible to trap a microscopic object and move it freely. If the object is stretched away from the focus by a certain external force, an equal yet opposite restoring force will be simultaneously generated. By measuring the restoring force, the external force on the object can also be obtained. Within a small distance range (~150 nm) near the focal center, the restoring force is linearly proportional to the displacement, which can be measured by the position sensitive detector in the OT system.120, 126 The calibration of the force can be performed based on the theory of thermal Brownian motion or the Stokes law.120, 127 It is also worth to note that the signal-to-noise ratio in OT measurements is relevant to the drag coefficients of the trapped objects. Smaller beads are better because they have smaller drag coefficients.120 1.2.2 SMFS Using OT To investigate the unfolding/folding of proteins, a dumbbell assay is often used, in which the target protein is sandwiched between two microbeads. DNA handles are frequently used as 18 linkers to avoid nonspecific interactions between the two beads. The linkage between the DNA handle and bead is often established through antigen-antibody interactions. One end of the DNA handle is modified with antigens, such as biotin (or digoxigenin), while the surface of the bead is coated with proteins, such as streptavidin (or anti-digoxigenin). The coupling between DNA handles and the target protein can be achieved through different strategies, including thiol coupling,15 click chemistry,128-129 protein tags130 and DNA oligonucleotide hybridization.131 Once the dumbbell is established, OT can be used to stretch and relax the target protein by manipulating the position of the trapped bead. The other bead is usually referred to as a stationary bead, fixed by a micropipette or another laser beam. In this way, OT can be used to stretch the DNA-protein-DNA construct to study the mechanical unfolding/folding of proteins (Figure 1.8). Figure 1.8 Schematics of the OT experiment to study a polyprotein GB1 (PDB: 2QMT) tetramer. The target protein is engineered with two cysteine residues at both the N- and C-termini. The bioconjugation between protein and DNA handles is achieved by thiol-maleimide interactions. The other ends of the DNA handles are modified with different antigens, which can be coupled with the antibody on the surface of each bead. One bead is fixed using a micropipette and the other is manipulated using a laser. The protein can then be mechanically unfolded and refolded by moving the trapped beads. To conduct an effective SMFS experiment using OT, two typical operating modes are used: constant-force mode and constant-velocity mode.132-133 19 1.2.2.1 Constant-Force Mode The constant-force mode is also known as the force-clamp mode. In constant-force mode, the protein of interest is held between two beads under a constant applied force. The applied force is kept at a set value using a force-feedback system. During the measurement, the end-to-end distance of the protein is recorded as a function of time. As shown in Figure 1.9, the unfolding and folding of a prion protein are probed via the constant-force mode. At a certain stretching force, if the native prion protein unfolds, the protein length extends to L1; if the unfolded prion protein refolds, the protein length shortens to L0. Figure 1.9 shows that the protein structure fluctuates between the native state and the unfolded state at equilibrium. Figure 1.9 A representative extension-time curve of a prion protein in constant-force mode. Abrupt jumps show that the prion protein unfolds/refolds in a two-state fashion. Image adapted with permission from National Academy of Sciences, USA, Copyright © 2012.134 For a given extension-time trajectory, a method that makes use of dwell-time can be used to extract the unfolding kinetics. Assuming the unfolding is a first-order reaction, the unfolding probability of the protein can be expressed as follows:135 𝑃𝑢(𝑡) = 1 − exp (−𝛼(𝐹) ∙ 𝑡) (1.3) where Pu(t) is the unfolding probability, α(F) is the unfolding rate constant at a given force F, and t is dwell-time, defined as the time interval when the protein stays in its native state. By taking the 20 derivative of probability with respect to time, the unfolding probability density is obtained and given by:135 𝑑𝑃𝑢(𝑡)𝑑𝑡= 𝛼(𝐹) ∙ exp (−𝛼(𝐹) ∙ 𝑡) (1.4) where dPu(t)/dt is the unfolding probability density that decays exponentially with dwell-time. By fitting the distribution of dwell-time to eq. (1.4), the unfolding rate constant at a given force F can be determined (Figure 1.10). Similarly, the folding rate constant at a given force can also be measured by following this method. The folding probability density can be expressed as follows: 𝑑𝑃𝑓(𝑡)𝑑𝑡= 𝛽(𝐹) ∙ exp (−𝛽(𝐹) ∙ 𝑡) (1.5) where dPf(t)/dt is the folding probability density, and β(F) is the folding rate constant at a bias force F. Here the dwell-time t is defined as the time interval when the protein stays in the unfolded state. As such, the force-dependent unfolding (or folding) rate constants can be measured. Fitting the force-dependent unfolding (or folding) rate constants to the Bell-Evans model can then yield the intrinsic unfolding (or folding) rate constant at zero force. 21 Figure 1.10 Unfolding probability density for all unfolding time intervals with a single exponential fit (black line; dotted lines represent 95% confidence intervals of the fit). Image adapted with permission from John Wiley and Sons, Copyright © 2017.136 1.2.2.2 Constant-Velocity Mode The constant-velocity mode, also known as the distance-ramp mode,117 is also frequently used in SMFS. In constant-velocity mode, the target protein is stretched and relaxed at a constant velocity. The applied force and the extension of protein length are simultaneously recorded. As shown in Figure 1.11, during the stretching process, the force rises nonlinearly as a function of extension. Once the protein is mechanically unfolded, the effective contour length of the protein increases, leading to an immediate force drop to a lower value. When the unfolded protein is relaxed to a sufficiently low force, protein refolding can occur, which is marked by a protein length shortening and a force jump to a higher value. 22 Figure 1.11 A representative force-extension curve showing the unfolding/folding of a prion protein. Dashed lines indicate WLC fits. Image adapted with permission from the National Academy of Sciences, USA, Copyright © 2015.137 The relationship between the force and the extension of a protein can be described using the worm-like chain (WLC) model138 of polymer elasticity: 𝐹(𝑥) =𝑘𝐵𝑇𝑝[14(1 −𝑥𝐿𝐶)−2−14+𝑥𝐿𝐶] (1.6) where F(x) is the entropic force at the extension of x, Lc is the contour length of polymer, kB is the Boltzmann constant, T is the absolute temperature and p is the persistence length of protein. The persistence length is defined as the distance over which the polymer retains its direction, suggesting a polymer’s bending stiffness or rigidity. The WLC model was developed to describe the behavior of semiflexible polymers, such as proteins, DNA and RNA.139 To perform the kinetics analysis in constant-velocity mode, a direct and model-free method proposed by Oesterhelt et al., is often used to extract the force-dependency of the unfolding and folding rate constants.140 The main idea of this method is to divide force-distance curves into many small time windows. Each time window is sufficiently small so that the force can be approximated 23 as constant. In this way, an experiment in constant-velocity mode can be effectively transformed into many constant-force experiments with each one at a different force. To obtain the unfolding (or folding) rate constant in constant-velocity mode, a different approach from fitting the distribution of dwell-time is used. Oesterhelt and coworkers showed that the unfolding rate constant at a given force can be directly calculated from the dwell-time and total number of unfolding events, given by: 𝛼(𝐹) =𝑁𝑢(𝐹)𝑀𝑢(𝐹)×∆𝑡 (1.7) where Nu(F) is the number of observed unfolding events at a given force F, Mu(F) is the number of data points taken at this force F, and ∆t is the time interval between two adjacent data points. The product of Mu(F)×∆t represents the total dwell-time of the force F acting on the native protein. Similarly, the folding rate constant at a given force can also be calculated using the Oesterhelt method, given by: 𝛽(𝐹) =𝑁𝑓(𝐹)𝑀𝑓(𝐹)×∆𝑡 (1.8) where Nf(F) is the total number of observed folding events at a given force F and Mf(F)×∆t represents the total dwell-time of the force F acting on the unfolded protein. The Oesterhelt method provides a convenient way to extract the force-dependent unfolding (or folding) rate constants from experiments operated in constant velocity mode. The Bell-Evans model is subsequently used to obtain the intrinsic unfolding (or folding) rate constant at zero force. 24 1.2.3 The Strengths and Limitations of OT Traditionally, AFM is the most widely used tool to investigate protein mechanical properties. By stretching a polyprotein with tandem repeated domains, a saw-tooth force-extension trajectory can be generated and used for further analysis. AFM is well-suited to monitor the unfolding process of proteins that exhibit high mechanical stability. However, the high stiffness of the commercial AFM cantilever (10-105 pN/nm) also brings limitations. A small change in cantilever deflection can give rise to a large increase in force, resulting in a limited force precision (~10 pN) and a high limit of detection (>10 pN).67 Moreover, the commercial AFM cantilever has high drag coefficients, leading to a relatively low signal-to-noise ratio in the measurements. Therefore, AFM often fails to detect the unfolding events of mechanically labile proteins, as well as metastable intermediate states during unfolding processes, as their unfolding forces are often below 10 pN. Additionally, it remains challenging to detect protein folding using AFM because folding often occurs at low force (<10 pN). Nevertheless, over the past ten years, the limiting resolution of AFM has been greatly improved, making it more versatile for the studies on protein unfolding/folding dynamics. For example, by tailoring commercial AFM cantilever geometry, Perkins and his colleagues have successfully improved the temporal resolution, force precision and force stability for AFM-based SFMS data.141-142 It is also important to note that many recent studies have achieved direct observation of protein folding using AFM.143-148 However, compared to commercial AFM, due to the high force stability and low trap stiffness (0.005-1 pN/nm),67, 115 OT is suitable to measure low forces (from 0.1 to 100 pN) with superior sensitivity (0.1 pN), thereby playing a significant role in revealing the folding of proteins. Despite the high versatility and precision provided by OT, OT also has some limitations.67 First, an optical trap lacks selectivity and exclusivity. Undesired beads and other small objects, 25 such as bacterial clusters and precipitated salt crystals can be captured. Therefore, delicate microfluidic chambers are frequently used to limit the beads from freely diffusing. Second, the low upper limit of the force range (~100 pN) makes it difficult to use OT to study mechanically stable proteins. For example, an SdrG B1 domain is a mechanically stable protein that requires over 2 nN of force to unfold.149 In that case, OT is a less appropriate tool to study unfolding behavior than AFM. Finally, the high-intensity laser can cause photodamage to the biomolecules and generate reactive oxygen species,150-151 which can affect the use of OT. Further improvements and efforts are still expected to expand the application of OT. 1.2.4 Review of Achievements on Protein Unfolding and Folding by OT Significant progress has been achieved in SMFS studies using OT. OT has elucidated the molecular mechanisms of a variety of biological processes, including stepwise movement of molecular motors,152-153 polymerase dynamics,154-155 packing of bacterial phage DNA,156 noncovalent interactions between ligands and receptors,157 etc.158 Here, in this section, some of the significant advances in the unfolding/folding of proteins are highlighted. 1.2.4.1 Directly Monitoring Mechanical Folding of Proteins by OT Pioneering investigations by OT were achieved by Bustamante et al.17 and Simmons et al.159, respectively in 1997. They studied the mechanical unfolding/folding of titin, a giant protein consisting of over 200 protein domains. Previous studies have suggested that titin is responsible for muscle ultrastructure and elasticity.18-19 Figure 1.12A shows a titin molecule that was stretched and relaxed at a constant velocity by OT. When pulled from the N- and C-termini, titin was observed to unfold at ~25 pN, leading to the lengthening of the protein. After all the titin domains unfolded, the unfolded titin was relaxed to allow for protein folding. At a low force of ~5 pN, the protein length started shortening, suggesting that unfolded titin started to refold. Although the 26 protein unfolding/folding observed in this experiment lacked fine details, this pioneering study opened the door to directly monitoring protein folding at the single-molecule level using OT. By using a dumbbell assay in the OT system, Bustamante and coworkers directly observed the folding of a protein (E. coli RNase H) (Figure 1.12B).15 At a low force of ~5.5 pN, the unfolded protein was observed to fold back into its native state with a sharp force jump accompanied by protein length shortening. Subsequent stretching generated similar unfolding patterns, suggesting that the protein had been successfully folded. Moreover, an on-pathway intermediate state in protein folding was clearly observed. Stretching this intermediate state resulted in protein unfolding at a lower force, suggesting that the partially folded protein was mechanically compliant. Figure 1.12 Direct observation of protein folding in the OT experiments. (A) Representative curve of stretching (red) and relaxing (blue) titin. Force-induced protein unfolding and folding can be observed at ~25 pN and ~5 pN. Arrows show the direction of stretching and relaxation. Image adapted with permission from the American Association for the Advancement of Science, Copyright © 1997.17 (B) Force-extension curves of E. coli RNase H. The protein is unfolded via a two-state process and folded via an intermediate state. Stretching the intermediate state generates unfolding at low force, implying that the intermediate state is mechanically unstable. Image adapted with permission from the American Association for the Advancement of Science, Copyright © 2005.15 Since, OT has enabled the direct observation of many protein folding processes. For example, Bustamante and his coworkers revealed the folding cooperativity of two subdomains of 27 the T4 lysozyme.160 By studying a Ca2+-binding protein, calmodulin, Rief et al. revealed a complex folding network involving four intermediate states.161-162 Woodside and his colleagues used OT to clearly characterize the folding of a dimeric prion protein.163-164 With the further development of SMFS techniques, more protein folding processes are expected to be fully elucidated. 1.2.4.2 Probing Misfolding and Chaperone-Assisted Folding of Proteins Some proteins might misfold and cannot perform their functions.165 Protein misfolding underlies a series of diseases, such as Alzheimer’s disease166 and Parkinson’s disease167. Understanding the molecular mechanism of protein misfolding is thus of great significance for pathology and therapy. Prion proteins consist of ~200 amino acid residues. Misfolded prion proteins have the ability to transmit misfolding onto other normal prion proteins. The misfolding of prion proteins can cause severe diseases, such as transmissible spongiform encephalopathies.168 OT provides a unique platform for investigating protein misfolding. Since nonnative proteins have a higher free energy, the misfolded proteins are often less stable than the native proteins. Woodside et al. have made significant advances in probing misfolding and aggregation of prion proteins using OT.22, 164 The key achievements are listed as follows: (1) They revealed that the correct folding of prion proteins is a two-state process. The short-lived intermediate states observed in the folding process are off-pathway, as the native state is only accessible from the unfolded state in the experiments.22 (2) Misfolding was observed to occur much more frequently than correct folding, suggesting that the prion protein samples a large number of conformations to reach the native state. Even though misfolding occurred frequently, the misfolded prion protein unfolded quickly due to the low mechanical stability of nonnative structures, ensuring efficient sampling.22 (3) The aggregation behavior of oligomers of prion proteins was also studied using OT.164 Their study revealed the 28 existence of nonnative interactions between adjacent prion proteins, leading to the formation of nonnative structures within the polyprotein. This observation suggested that the prion protein was prone to aggregation. So far, many proteins have been observed to misfold in OT experiments.169-172 Protein misfolding is a ubiquitous phenomenon in nature; however, in most cases, proteins are correctly folded into their native states.173 The key might lie in the fact that the cell provides an environment to ensure correct and productive protein folding. Many large proteins that have complex structures fold from nascently synthesized polypeptides on the ribosome.78, 174 Chaperones are also required in the folding of these proteins.175-177 Even though protein misfolding may occur in the cellular environment, there usually exists a degradation mechanism that quickly digests misfolded proteins.178 A series of OT studies have investigated protein folding in the presence of ribosomes and chaperones. 79, 179-182 In particular, Kaiser et al. examined the folding behavior of a multi-domain protein named elongation factor G (EF-G) in the presence of ribosomes and chaperones using OT (Figure 1.13).79, 179 EF-G is a large protein composed of five different domains. To study the co-translational folding of EF-G, they used an in vitro translation system to prepare a series of ribosome-nascent chain complexes, which were used in OT experiments. The results suggested that ribosomes can help prevent misfolding by destabilizing misfolded structures and reducing the folding rate. As such, misfolding among domains was significantly reduced. The effect of the chaperone trigger factor (TF) was also investigated in this study. TF is a ribosome-binding chaperone that binds the ribosome near the polypeptide exit tunnel.183 It was found that TF helped avoid protein misfolding and protected the folded domains against denaturation. Their study on 29 EF-G revealed that the ribosome and chaperone together guide protein folding into the native structure. Figure 1.13 Protein EF-G and experimental schematics of OT to stretch the ribosome-nascent chain complex. Protein EF-G was expressed in vitro and stalled at a designated residue. The N-terminus of the protein and ribosome was tethered by DNA handles. The effect of TF was also investigated in this study. Image adapted with permission from Elsevier, Copyright © 2019.79 1.2.4.3 Combining Fluorescence Spectroscopy and OT In SMFS studies, we can infer whether protein unfolding (or folding) occurs by measuring force vs distance (or extension vs time). However, protein unfolding/folding cannot be directly visualized. Technological advances have successfully combined OT and fluorescence spectroscopy, providing highly complementary information (i.e., force and fluorescence) associated with protein unfolding/folding.158, 184-187 Such studies require that the target protein is fluorescent and that the fluorescence can be clearly detected. Green fluorescent protein (GFP) contains an intrinsic chromophore and is a good candidate for this type of study. Rief et al. described a direct relationship between the fluorescence and structural dynamics of GFP using OT (Figure 1.14A).186 In the OT experiments, force and fluorescence were simultaneously recorded to monitor the unfolding and folding of GFP. Their study showed that fluorescence was quenched at the early stage of unfolding, suggesting that the 30 completely folded structure is necessary for GFP to exhibit fluorescence. This work not only characterized the mechanical properties of GFP but also revealed the relationship between protein structure and fluorescence. For proteins that are not intrinsically fluorescent, a chemical modification method is often used to modify the protein with fluorophores, e.g., Förster resonance energy transfer (FRET) pairs. Ha et al. studied spider silk flagelliform protein using a combination of OT and single-molecule FRET (Figure 1.14B).187 The cyanine dyes Cy3 and Cy5 were chemically modified at the N- and C-termini of the protein. The end-to-end distance of the protein and the FRET signal between Cy3 and Cy5 were simultaneously monitored. At a high force, the distance between Cy3 and Cy5 is large, leading to a low FRET; at a low force, the distance between Cy3 and Cy5 is shortened, resulting in a high FRET. Therefore, force and FRET are highly correlated. By characterizing the relationship between force and FRET, this study suggested that spider silk flagelliform could potentially be developed as a force sensor. 31 Figure 1.14 Combining fluorescence spectroscopy and OT to study protein unfolding and folding. (A) Mechanical unfolding and folding of GFP lead to an immediate change in force and fluorescence. The fluorescence quenches immediately when GFP unfolds, and recovers after a complete refolding of GFP. Image adapted with permission from the National Academy of Sciences, USA, Copyright © 2017.186 (B) Stretching-relaxing spider silk flagelliform protein leads to a change in FRET. Stretching separates the Cy3-Cy5 pair, leading to a low FRET; relaxation brings the pair near to each other, leading to a high FRET. Image adapted with permission from the American Chemical Society, Copyright © 2016.187 This combination of OT and fluorescence spectroscopy has also been applied to probe a wide variety of biological processes, including the unfolding of DNA hairpins184, RNA hairpins188, and dissociation of DNA-protein complexes189. Nevertheless, further technological progress is still needed, for example, to increase the fluorescence intensity and circumvent photobleaching.158 1.3 The Aim of This Thesis OT is one of the most powerful techniques used to investigate the mechanical unfolding/folding of proteins at the single-molecule level. Inspired by pioneering work in the field, 32 here I intend to use OT to study the unfolding/folding of two classes of proteins, namely knotted/slipknotted proteins and Ca2+-binding proteins. Previous SMFS studies have characterized the unfolding/folding of the shallow slipknotted protein AFV3-109.190-192 Results revealed that upon stretching, AFV3-109 can easily be untied into an unfolded polypeptide chain; upon relaxation, AFV3-109 rapidly folds back into its native state. However, the unfolding/folding of a slipknotted protein with a long threaded loop is poorly understood. The purpose of chapter 2 is to study the mechanical unfolding and folding of the complex slipknotted protein PADC. PADC is a slipknotted protein with a long threaded loop. When stretched from the N- and C-termini, whether the slipknot can be completely untied remains unknown. Additionally, it is also worth studying whether PADC refolds back into its native state upon relaxation. Our investigations will demonstrate the complexity of the mechanical unfolding-refolding of a slipknotted protein with a long threaded loop. Previous studies have suggested that knotting is critical in the folding of knotted proteins.193-195 However, this knotting process has rarely been investigated at the single-molecule level.170, 196 The goal of chapter 3 is to mechanically tighten, untie and retie the knotted protein TrmD using OT. We will explore whether the knotted structure is critical for maintaining the stability of the protein, and whether knotting is the rate-limiting step during the folding process. To understand the mechanical features that are responsible for efficient translocation of RTX domains, in chapter 4, we investigate the mechanical unfolding and folding of a Ca2+-binding protein, RTX block V. We want to reveal detailed conformational changes of the protein during the unfolding/folding process. Our results will provide mechanistic insights into the translocation mechanism of RTX block V. 33 To examine whether the translocation of other RTX domains occurs via a similar mechanism, in chapter 5, we study another RTX domain, RTX block IV, which is adjacent to the N-terminus of RTX block V. The mechanical unfolding/folding of RTX block IV will be studied and compared with that of RTX block V. We will also study whether the mechanical stability of RTX block V is affected by RTX block IV. In chapter 6, the conclusions obtained from the above experimental efforts will be summarized, and several potential future studies on the unfolding/folding of knotted/slipknotted proteins and Ca2+-binding proteins will be discussed. 34 Chapter 2: Mechanical Unfolding and Folding of a Complex Slipknotted Protein Probed by Using Optical Tweezers 2.1 Synopsis Knotted and slipknotted proteins are topologically complex. Understanding their unfolding and folding mechanism has attracted considerable interest. In this chapter, we combine protein engineering, OT, and steered molecular dynamics (SMD) simulations to investigate the mechanical unfolding and folding of a complex slipknotted protein PADC. In the slipknotted structure, PADC contains a long threaded loop (85 residues), which is almost twice the size of the knotting loop. When stretched from its N- and C-termini, the majority of PADC can be readily unfolded in a two-state manner, and the slipknotted structure was untied. A small percentage of PADC followed a three-state unfolding pathway involving the formation of an intermediate state. These unfolding intermediate states showed a broad distribution of contour length increments, suggesting that they did not have a well-defined specific structure. SMD simulations revealed the main free energy barrier to the unfolding of PADC and suggested that the unfolding intermediate states may originate from the friction of polypeptide chain sliding, during the process of pulling the threaded loop out of the knotting loop. Upon relaxation, a small percentage of the unfolded and untied polypeptide chain can refold back to its native slipknotted conformation, but a large fraction can only reach a misfolded state. Our results revealed the complexity of the mechanical unfolding and refolding of the slipknotted protein with a long threaded loop. 35 2.2 Introduction Proteins are linear polypeptide chains, which are synthesized on the ribosome and fold into unique 3D native structures.39, 165 If stretched from the N- and C-termini, most proteins will be stretched into an extended linear chain without any knots. However, a few well-tied knots, which are deeply embedded in the protein structures, have been discovered in proteins since the late 1990s.83, 86-87 Thanks to the advance in bioinformatics over the past two decades, a small yet significant number of proteins have been identified as knotted or slipknotted proteins, accounting for ~2% of the proteins in the PDB.90, 197 Knotted and slipknotted conformations are surprising features for proteins, when considering the complexity of the protein folding problem itself. Adding another layer of topological complexity to protein folding raises the question how such knotted and slipknotted proteins overcome these topological barriers to fold into their native conformations.94, 198 In addition, the functional and structural significance of such knotted and slipknotted proteins remains to be fully elucidated. Both experimental,170, 193-194, 199 including ensemble as well as single-molecule studies, and simulations studies94, 198 have provided invaluable insights into the folding mechanism of knotted proteins. The folding of knotted proteins was found to be generally slow and involve overcoming a high topological barrier.198 The complexity of the free energy landscape for some knotted proteins has been probed.170, 200-201 Molecular dynamics simulations revealed different possible folding mechanisms for knotted proteins, including the threading, slipknotting, mouse trapping, as well as folding on the ribosome mechanisms.94, 202-204 Different from knotted proteins, slipknotted proteins contain knotted subchains, but their overall backbone chain is unknotted.197 In a slipknotted protein, a threaded loop (also called slipknot loop) is threaded through the knotting loop (also called knot core) (Figure 2.1A). When 36 stretched from its N- and C-termini, the slipknotted protein could be untied into a linear polypeptide chain, in a way similar to untying shoelaces. In contrast to the folding of knotted proteins in which the formation of the knot conformation is often the rate-limiting step, the folding of slipknotted proteins is determined by the relative length of the threaded loop and the knotting loop.198 How such slipknotted proteins fold and what functional significance of a slipknotted structure possesses are both important questions. 37 Figure 2.1 Stretching and relaxing a slipknotted protein by using OT. (A) The 3D structure of the slipknotted protein PADC (PDB: 2QQD) (residues 7-165). PADC is a α/β protein and shows a four-layer αββα sandwich structure. The knotting loop (colored in red) consists of amino acid residues 19-75, involving 1 α helix and 4 β strands. Residues 76-161, involving 1 α helix and 5 β strands, form the threaded loop (colored in yellow) through the knotting loop. The sequences colored in blue are slipknot tails. In the OT experiments, the slipknotted protein is stretched from its N- and C- termini. (B) Schematics of the MiniTweezers OT setup. The streptavidin-coated polystyrene bead (1 μm) is held by a glass micropipette via suction, and the anti-digoxigenin-coated bead (2 μm), which is sparsely coated with the protein-DNA sample, is trapped in the laser trap. The two beads are brought into contact with each other to establish the dumbbell for stretching. The protein chimera NuG2-PADC-NuG2 is flanked by two dsDNA handles, which are 802 bp and 558 bp long, respectively. NuG2 serves as a fingerprint for identifying single-molecule stretching events and an internal caliper. In their pioneering work, Sulkowska and co-workers used the two slipknotted proteins AFV3-109 and thymidine kinase, which have very different sized threaded loops, as model 38 systems to investigate the complex unfolding and folding mechanism of slipknotted proteins via coarse grained molecular dynamics simulations.198, 205 Using the slipknotted protein AFV3-109 as a model system, He et al. have investigated the mechanical unfolding and refolding of slipknotted proteins.190-192 In the AFM experiments, they found that upon stretching, the slipknotted conformation of AFV3-109 can be untied and unfolded into an extended and unknotted polypeptide chain.190 In the OT experiments, they also found that upon relaxation, AFV3-109 can fold back to its native slipknotted conformation rapidly at low forces.192 The fast folding of AFV3-109 implied that the folding of this slipknotted protein does not involve a high topological barrier. However, since AFV3-109 has a relatively short threaded loop (~20 residues), it remains unknown whether slipknotted proteins that have a much longer threaded loop will fold in a similar fashion. To address this question, here we used a slipknotted protein PADC, which has a much longer threaded loop than the knotting loop, as a model system to investigate its mechanical unfolding and refolding behaviors via OT. PADC, an enzyme catalyzing the reaction of polyamine biosynthesis in archaebacteria and plants,93 is a slipknotted α/β protein that contains a long threaded loop (Figure 2.1A). The knotting loop (colored in red) consists of residues 19-75, and the threaded loop (colored in yellow) consists of residues 76-161. The threaded loop of PADC (85 residues) is much longer than that of AFV3-109 (~20 residues), providing an ideal model system for studying the unfolding and folding mechanism of slipknotted proteins with long threaded loops. Here, we combine protein engineering and the OT technique to investigate the unfolding and folding of PADC. Our results showed that upon stretching from its N- and C-termini, PADC could be untied and unraveled to a linear polypeptide chain via multiple unfolding pathways, including two-state and multistate pathways involving intermediate states. Upon relaxation of the untied and unfolded polypeptide, 39 a small percentage of unfolded PADC could refold back into its native state. Distinct refolding pathways were observed, suggesting the complexity during the refolding process of the slipknotted structure. 2.3 Results and Discussion 2.3.1 Stretching PADC Leads to the Untying of the Slipknot and Complete Unfolding of PADC. To investigate the mechanical unfolding and folding of PADC, we constructed the DNA-NuG2-PADC-NuG2-DNA hybrid construct for single-molecule OT experiments, where PADC is flanked by two NuG2 fingerprint domains. The mechanical unfolding and folding of NuG2, which has been studied in detail in our previous work,206 is characterized by a contour length increment (ΔLc) of ~18 nm. In this study, NuG2 domains serve as fingerprints in the construct NuG2-PADC-NuG2 and allow for the identification of the single-molecule stretching event of NuG2-PADC-NuG2 and the mechanical unfolding/folding signature of PADC. Stretching the DNA-NuG2-PADC-NuG2-DNA construct resulted in characteristic sawtooth-like force-distance curves. As shown in Figure 2.2A, the force-distance curves of the full length NuG2-PADC-NuG2 show three unfolding force peaks: two of them (colored in red) occurred at relatively higher forces with a shorter ΔLc, while the third one (colored in black) occurred at a relatively lower force with a much longer ΔLc. Fitting the force-extension of the unfolding events to the WLC model (Section 1.2.2.2) of polymer elasticity revealed that the two unfolding events in red show a ΔLc of ~18 nm, characteristic of NuG2, so these two unfolding events can be attributed to the unfolding of two fingerprint domains of NuG2. Accordingly, the unfolding event colored in black can be readily attributed to the unfolding of the slipknotted protein PADC. Fitting the WLC model to the length increase at different forces for the unfolding events 40 of PADC allowed us to measure an average ΔLc of 56 ± 3 nm (Figure 2.2B). The folded structure of PADC contains 159 amino acid residues (residues 7-165). The untying the slipknotted structure and the subsequent complete unraveling of PADC will result in a ΔLc of 56.0 nm (159 aa × 0.36 nm/aa – 1.2 nm = 56.0 nm, where 1.2 nm is the distance between N- and C-termini), in good agreement with the experimentally determined ΔLc. This result suggests that the stretching of PADC leads to the untying of the slipknot and unfolding of the folded structure. 41 Figure 2.2 Stretching PADC from its N- and C-termini leads to the untying of the slipknot and the complete unfolding of PADC. (A) Representative force-distance curves of NuG2-PADC-NuG2 at a pulling speed of 50 nm/s. The unfolding events colored in red correspond to the mechanical unfolding of the fingerprint domain NuG2. The unfolding events colored in black correspond to the unfolding of PADC. In the majority of the unfolding trajectories of PADC, the unfolding occurs in a two-state fashion without the accumulation of any intermediate states. (B) Force-extension relationships of the unfolding of PADC. Solid lines are the WLC fits to the force-extension data measured from the force-distance curves. The WLC fits yielded a persistence length of 0.8 nm and ΔLc of 56 ± 3 nm. (C) Unfolding force histogram of the two-state unfolding of PADC. Inset: force dependency of the unfolding rate constants. This force-dependency was obtained from the force-distance curves using the method proposed by Oesterhelt et al. Fitting the Bell-Evans model to the experimental data measures the intrinsic unfolding rate constant α0 of 0.1 s-1 and the unfolding distance Δxu of 0.3 nm. 42 2.3.2 The Mechanical Unfolding of PADC Follows Multiple Pathways. In most of the unfolding trajectories of PADC (83%), the unfolding occurs in an all-or-none fashion without the accumulation of any unfolding intermediate states, suggesting that the unfolding of PADC occurs in a two-state fashion. Such two-state unfolding events occurred over a relatively broad range of forces, with an average unfolding force of ~15 pN (Figure 2.2C). We used the method proposed by Oesterhelt et al.140 to convert the measured unfolding forces to force-dependent unfolding rates (Figure 2.2C, inset). Fitting the experimental data using the Bell-Evans model74, 207-208 yielded the intrinsic unfolding rate constant α0 of 0.1 s-1 at zero force and the unfolding distance Δxu of 0.3 nm. This small Δxu suggested that the structure of the slipknotted protein PADC is rather brittle. In addition to the two-state unfolding trajectories, about 17% of unfolding trajectories of PADC occurred involving the formation of one, or two (in rare cases) unfolding intermediate states (Figure 2.3A). The sum of the two contour length increments ΔLc1 + ΔLc2 is equal to ~56 nm, indicating that the unfolding of PADC occurred in two distinct steps involving the formation of one unfolding intermediate state. These results revealed the complexity of the mechanical unfolding process of this slipknotted protein. It is also of note that ΔLc1 (and ΔLc2) showed a broad distribution, suggesting that the unfolding intermediate state is not well-defined and might show a wide range of structures (Figure 2.3B). This observation is quite different from the results obtained for most protein unfolding intermediate states observed in SMFS experiments,15, 161, 209 where the unfolding intermediate state is well-defined along their mechanical unfolding pathways and shows a defined ΔLc. 43 Figure 2.3 Three-state unfolding of PADC. (A) Force-distance curves of PADC showing three-state unfolding behaviors. In ~17% trajectories, the unfolding of PADC occurred in a three-state fashion, involving the formation of one unfolding intermediate state. The sum of ΔLc1 and ΔLc2 is ~56 nm, corresponding to the complete unfolding of PADC. (B) The histogram of ΔLc1 of the three-state unfolding of PADC. ΔLc1 shows a broad distribution, ranging from ~10 to ~50 nm, indicating that the unfolding intermediate state is not well-defined. 2.3.3 SMD Simulations of Mechanical Unfolding of PADC. To investigate the molecular mechanism of the mechanical unfolding of PADC and understand the molecular origin of the broad distribution of unfolding intermediate states in the three-state unfolding pathways, we carried out SMD simulations210-211 of the mechanical unfolding of PADC using constant-velocity mode. PADC is a slipknotted protein with a long threaded loop. On the one hand, stretching PADC from its two termini tends to pull the threaded loop out of the knotting loop, leading to the untying of the slipknot; on the other hand, the stretching can also lead to the contraction of the knotting loop, leading to the tightening of the slipknot. The competition between these two processes will determine the unfolding trajectories of PADC. 44 Figure 2.4A shows two representative force-extension curves from constant velocity simulation trajectories. Upon stretching, the N-terminal α helix is first unraveled, accompanied by the alignment of the N, C-termini with the pulling force. The first major barrier encountered during the stretching of PADC originates from the β sheet formed by β strands β1, β9 and β5 (Figure 2.1A). Disruption of the interactions between β1 and β9 resulted in a major unfolding force peak in the force-extension curve. During this process, half of the αββα sandwich (including β1, β5, β9 and α1) structure was disrupted, and β1 was pulled out of the knotting loop. After overcoming this major unfolding barrier, the rest of the protein structure was gradually unraveled and extended. During the subsequent stretching process, the threaded loop was gradually pulled out of the knotting loop, a process involving the sliding of some part of the polypeptide chain along each other. Such sliding generated considerable friction within the polypeptide chain. Overcoming the friction led to significant force peaks in the resultant force-extension curves. The location of “such barriers” is not well-defined but can occur over a broad range of distances (Figure 2.4B). Such additional barriers/unfolding force peaks are qualitatively similar to the unfolding intermediate states observed in the OT experiments. It is worth noting that, during the stretching process, the knotting loop contraction is accompanied by the threaded loop being gradually pulled out of the knotting loop, leading to a much smaller and somewhat tightened slipknot at a larger end-to-end distance of PADC (Figure 2.4A). Further pulling led to the eventual untying of the slipknotted structure. This observation has some resemblance with the tightened slipknot intermediate state observed in the coarse-grained molecular dynamics simulations of thymidine kinase that has a much longer threaded loop.205 However, different from the tightened slipknot intermediate state observed in thymidine kinase, 45 the smaller slipknot observed for PADC is much looser and can be untied readily without requiring significantly higher forces. Figure 2.4 SMD simulations of the mechanical unfolding of PADC. (A) Representative force-extension curves of PADC in the constant-velocity SMD simulations. Snapshots of the PADC structure are also shown. The first major unfolding event of PADC corresponds to the disruption of the interactions between β1 and β9. After this event, a large portion of the folded structure unfolds, and β1 was pulled out of the knotting loop. During stretching, the knotting loop shrank, and the threaded loop got shorter. The threaded loop was pulled out of the knotting loop at long extensions, leading to untying the slipknot. (B) Rupture force peaks were observed at different at different extensions. These force peaks mainly originated from chain frictions due to polypeptide chain sliding when the threaded loop was gradually pulled out of the knotting loop. 46 2.3.4 Mechanical Unfolding Mechanism of PADC. Based on the SMD simulations results, it is now possible to gain some insights into the possible mechanical unfolding mechanism of PADC under a stretching force. In the OT experiments, the unfolding of most PADC followed a simple two-state behavior and showed a ΔLc that corresponds to the untying of the slipknot and the complete unfolding of PADC. The major barrier likely corresponds to the rupturing of interactions between β strands 1 and 9. After overcoming this mechanical unfolding energy barrier, PADC unfolds cooperatively and loses its folded tertiary structure. Finally, the unfolded polypeptide chain behaved as a random coil, in which the slipknot was untied quickly. This unfolding mechanism is somewhat different from the SMD simulations, where the slipknot structure persisted during the stretching and only untied toward the end of the pulling trajectories. This difference is likely due to the large differences in the time scale of the OT experiments and SMD simulations (milliseconds versus nanoseconds). The three-state unfolding for PADC in the OT experiments shares a more qualitative similarity with SMD simulations. Compared with SMD simulations, the unfolding events associated with the unfolding intermediate states in the OT experiments are likely arising from friction due to polypeptide chain sliding when the threaded loop was pulled out of the knotting loop. Although the slipknot was eventually untied in such three-state unfolding pathways, it remains unclear if the slipknot was untied during the first (ΔLc1) or the second step (ΔLc2). It is also important to note that the unfolding of PADC does not involve the formation of a true tightened slipknot intermediate state, as predicted for the large slipknotted protein thymidine kinase.205 In our SMD simulations, a contracted slipknot was present in most trajectories of PADC. However, this contracted slipknot structure was loose enough to be untied easily so that it did not cause jamming. This is distinct from the tightened slipknot intermediate state in the thymidine 47 kinase. Nonetheless, our results confirmed the possibility of forming a tightened slipknot intermediate state during the mechanical unfolding of a slipknotted protein with a long threaded loop, and suggested that the formation of the tightened slipknot intermediate state may depend on the specific slipknotted structure as well as the pulling velocity. Moreover, the unfolding mechanism of PADC also contrasts with that of AFV3-109. PADC has a long threaded loop (85 residues), and a relatively short knotting loop (57 residues), while AFV3-109 has a short threaded loop of only 20 residues but a longer knotting loop (70 residues). The large difference in the size of the threaded loop in these two proteins provides an opportunity to gain additional insights into the unfolding mechanism of slipknotted proteins. The mechanical unfolding of slipknotted protein depends on the competition between the contraction of the knotting loop and pulling the threaded loop out of the knotting loop, as evidenced in the SMD simulations on AFV3-109 and PADC. In the mechanical unfolding of AFV3-109, the short threaded loop was always pulled out of the knotting loop first, while the long knotting loop did not contract.190 The unfolding of PADC showed a different behavior. In the two-state unfolding process, PADC unfolded in a cooperative fashion, leading to the untying of the slipknot and complete unfolding of PADC. In the three-state unfolding, the contraction of the knotting loop was observed to be accompanied by the threaded loop being gradually pulled out. This difference reflected the impact of the length of the threaded loop on the mechanical unfolding behaviors of slipknotted proteins and suggested the possibility for forming a tightened slipknot intermediate state. 48 2.3.5 Untied and Unfolded PADC Can Fold Back into Its Native Slipknot Conformation: Complexity in the Folding of PADC. After mechanical unfolding of PADC, the slipknotted PADC is converted to an unknotted and unfolded polypeptide chain. Relaxing this untied and unfolded polypeptide chain will allow us to investigate whether an unfolded polypeptide chain folds into its native slipknotted conformation. Upon relaxation, we observed a force jump at ~3 pN accompanied by the shortening of the polypeptide chain to the original length of the folded slipknotted protein. Subsequent stretching revealed that the unfolding signatures of the refolded protein are identical to those of the slipknotted native state (unfolding force at ~15 pN and ΔLc of 56 nm), strongly suggesting that the unfolded polypeptide chain had refolded back into its native slipknotted conformation. However, the refolding of PADC showed a very complex picture. Among the 36 molecules we studied, only 9 molecules (25%) can readily fold into the slipknotted native conformation during the stretching-relaxation cycles (referred as successful folders). Figure 2.5A shows the unfolding-refolding cycles for one such molecule, where the refolding occurred in a two-state fashion without the accumulation of intermediate states. For these molecules, the refolding forces showed a narrow distribution with an average value of ~3 pN (Figure 2.5B). Using the method proposed by Oesterhelt et al.,140 we measured the relationship between the folding rate constant and force. Fitting the data using the Bell-Evans model yielded the intrinsic folding rate constant at zero force β0 of 81 s-1 (Figure 2.5B, inset). These results indicated that these unfolded PADC molecules can readily refold into the native slipknotted conformation, and the folding does not involve a high topological barrier. This folding behavior of PADC is similar to that of the slipknotted protein AFV3-109 with a short threaded loop, whose folding is rapid and robust.192 49 Figure 2.5 Unfolded PADC can fold back to its native slipknotted state. (A) Representative stretching-relaxation curves of PADC. Unfolded PADC was able to fold back to its native state in a two-state fashion. Consecutive force-distance curves from one PADC molecule show the successful protein folding to the native state. (B) The folding force histogram of PADC. The folding force is narrowly distributed at ~3 pN. Inset shows the force dependency of the folding rate constants obtained using the Oesterhelt method. Fitting the data to the Bell-Evans model revealed the intrinsic folding rate constant β0 of 81 s-1. (C) Representative force-distance curves of misfolding of PADC (stretching curves are colored in red and relaxation curves in blue). The first and second cycles are offset in distance. After the first successful unfolding of PADC, PADC folded into a mechanically compliant conformation, which has a similar length to the native conformation. The unfolding of this “misfolded” state resulted in a hump-like feature, and the subsequent relaxation curve showed a similar behavior. (D) Superposition of 10 consecutive force-distance curves (in grey) of the misfolding PADC. The average (in black) of these 10 curves clearly shows a hump-like feature. Red lines are pseudo WLC fits to the force-distance curves and are primarily used to indicate the two contour lengths, and the blue line is the derivative of the average of the force-distance curves. 50 In contrast, in the other 27 molecules, more than 50% of trajectories did not result in a successful folding event into the native state of PADC (referred as unsuccessful folders). Instead, most of the trajectories resulted in a recoiling of the polypeptide chain. Subsequent stretching did not lead to a characteristic unfolding event of the slipknotted protein PADC. For example, Figure 2.5C shows two cycles of stretching-relaxation curves of NuG2-PADC-NuG2. In the first cycle, a clear unfolding event of PADC with ΔLc of ~56 nm was observed. Upon relaxation, only elastic recoiling was observed. In the second cycle, stretching the molecule to higher forces did not lead to the typical PADC unfolding event, only the unfolding events of two NuG2 domains were observed and the relaxation curve showed typical refolding events of NuG2 domains. These “refolding” and subsequent “unfolding” curves of PADC could not be well described by the WLC model, but showed an evident deviation. A hump-like feature was present in the force-distance curves at ~2 pN, and this feature is clear in the averaged curve from ten individual curves (Figure 2.5D). Such a hump feature is indicative of a conformational change of the protein and has been observed in the unfolding/folding of villin headpiece,212 as well as conformational changes in polymers, such as polyethylene glycol and amylose.213-214 Although the nature of this hump-like feature in PADC remains unknown, it is clear that the unfolded PADC managed to refold into a conformation that is different from the native slipknotted conformation, a misfolded state. This misfolded state has a similar length to the native slipknotted conformation, but it is unclear whether the slipknot has formed. It is also of note that, starting from the unfolded PADC, 3 molecules out of the 9 successful folders always folded back into the native conformations. For these molecules, the folding appeared as a two-state event with a clear force jump at the low force regime, suggesting a fast refolding into the native slipknotted conformation. On the other hand, 13 molecules out of the 27 51 unsuccessful folders always folded back into the misfolded states. Allowing these molecules to fold at low forces for some time (up to 1 minute) did not appear to affect the folding pathway. It seems that the folding pathway is defined by the “starting” state of the unfolded polypeptide, and the unfolded polypeptide chain can assume a folding-prone state or a misfolding-prone state. However, the origin of this behavior remains unknown. Similar pathway switching behaviors were also observed in the folding of GFP,186 where some unfolded GFP can readily fold back to its native conformation, while others cannot in the time scale of 5 min. It is interesting to note that PADC contains 11 proline residues, and the proline isomerization might be a possible reason. It remains to be examined whether proline isomerization is involved in this process. This complex folding behavior is in sharp contrast to the rapid and robust folding of the shallow slipknotted protein AFV3-109, and clearly indicates the complexity in the folding of PADC with a long threaded loop. It is worth noting that the folding of slipknotted proteins might be related to the ratio of the length of the threaded loop and the knotting loop. A successful folding requires the formation of the knotting loop and threading of the threaded loop. The length of the threaded loop in PADC is much longer than the size of the knotting loop, which adds additional topological difficulty for correct folding. Moreover, in the OT experiments, since the N- and C-termini are tethered to the polystyrene beads and cannot move freely, this pulling geometry may further constrain the folding and contribute to the low efficiency of the folding of PADC. However, the detailed folding mechanism of PADC remains largely inaccessible in the current experiments. Coarse-grained molecular dynamics (MD) simulations will be required to provide further insight. Moreover, the low efficiency of the folding of PADC in our OT experiments also raises the question on how PADC folds efficiently in vivo, which is essential for the biosynthesis of 52 polyamines in archaebacteria and plants.93 It has long been recognized that protein folding in vitro may be different from that in vivo, especially for large, multidomain proteins.215-216 The impact of chaperones and cotranslational folding, where folding occurs concurrently with protein synthesis on the ribosome, has been well reported.79, 215-216 Thus, it will be important to investigate such factors for the folding of slipknotted proteins. It has been demonstrated that the folding of some knotted proteins is assisted by chaperones.217 Whether the folding of PADC requires chaperones in vivo is certainly worth exploring. In addition, it is also important to investigate the effect of cotranslational folding on the folding reaction of PADC. Recent computation studies on the folding of knotted protein YibK proposed that cotranslational folding on the ribosome could facilitate the formation of the trefoil knots.202, 204 If this mechanism is general, the formation of slipknots may be accelerated for the folding of such slipknotted proteins with long threaded loops. To verify such mechanisms, experiments that directly probe the folding kinetics of slipknotted proteins on the ribosome will be required. 2.4 Conclusions By combining OT and SMD simulations, we investigated the mechanical unfolding and folding of the slipknotted protein PADC with a long threaded loop. When stretched from its N- and C-termini, PADC can be mechanically unfolded and untied into a linear polypeptide chain in a two-state fashion. In rare occasions, three-state unfolding was observed involving an intermediate state, which is not well-defined but shows a broad range of structures. Such behavior likely results from the friction of polypeptide chain sliding when the threaded loop is pulled out of the knotting loop. Moreover, the unfolded and untied PADC can refold back into the native state when the polypeptide chain is relaxed. The folding of PADC also showed complex behaviors involving misfolding. Our studies revealed the complexity associated with a slipknotted protein 53 that has a long threaded loop, and demonstrated the necessity of combining SMFS with MD simulations to understand the folding and unfolding mechanism of such complex proteins. 2.5 Experimental Section 2.5.1 Protein Engineering. The gene encoding PADC with restriction sites (5’ BamHI and 3’ KpnI) was custom synthesized (GeneScript). To circumvent undesired coupling to the designed double-strand DNA (dsDNA) handles during the coupling reactions, the cysteine residues in the wt PADC were mutated to serine create a cysteine-free mutant. To avoid the internal autoserinolysis that would break the protein backbone, Ser53 was mutated to Ala.93 Using well-established molecular biology protocols, we engineered NuG2-PADC-NuG2, where the well-characterized NuG2 serves a fingerprint domain.144, 206, 218 The PADC gene was first subcloned into a pUC19 vector via BamHI and KpnI restriction sites; two genes encoding NuG2 domain were then inserted into the pUC19/PADC plasmid by two alternating enzyme digestion and ligation steps to construct pUC19/HindIII-NuG2-BamHI-PADC-KpnI-NuG2-EcoRI. NuG2 with the corresponding sticky ends was generated by the polymerase chain reaction (PCR) amplification technique. Next, NuG2-PADC-NuG2 was subcloned into the expressing vector, a modified pET-22b (+) vector, via HindIII and EcoRI restriction sites. The two restriction sites on the modified pET-22b (+) vector were created by completely removing the MCS region by PCR amplification, and the inset with the sticky ends digested by HindIII and EcoRI restriction enzymes could be ligated into the modified pET-22b (+) vector within the reading frame. The designed expression vector also contains one cysteine residue codon before the HindIII restriction site and behind the his-tag tail, respectively, enabling the dsDNA handles to be coupled to the N- and C-termini of the whole construct. The protein sequences of PADC and NuG2 are given in Appendix. 54 The engineered protein was over expressed in Escherichia coli strain BL21 (DE3) at 37 °C in 250 mL 2.5% LB media containing 100 mg/L ampicillin. 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG, Thermo Fisher Scientific, Waltham, MA) was added to induce the protein overexpression at an optical density around 0.8. The protein overexpression was subsequently allowed to continue for 4 hours at 37 °C. The bacteria cell pellets were harvested by centrifugation at 5,000 rpm at 4 °C for 10 minutes and resuspended in 10 mL phosphate buffered saline (PBS, pH 7.4) buffer. 10 μL protease inhibitor cocktail (SIGMA-ALDRICH, St. Louis, MO), 50 μL 100 mg/mL lysozyme from egg white (SIGMA-ALDRICH, St. Louis, MO), 1 mL 10% (w/v) Triton X-100 (VWR, Tualatin, OR), 50 μL 1 mg/mL DNase I (SIGMA-ALDRICH, St. Louis, MO) and 50 μL 1 mg/mL RNase A (Bio Basic Canada Inc, Markham, ON) were added for cell lysis. The lysis reaction was kept at 4 °C for 40 minutes. The supernatant containing the target protein was isolated by centrifugation at 10,000 rpm at 4 °C for 1 hour, and the protein was purified by Co2+ affinity column with a TALON His-tag purification kit (TaKaRa Bio USA Inc, Mountain View, CA). The protein was eluted and stored in elution buffer (PBS, 300 mM NaCl, 250 mM imidazole). The purified protein was at a concentration of ~1.0 mg/mL and stored at -20 °C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to confirm the molecular weight of the polyprotein (Figure 2.6). 55 Figure 2.6 Coomassie blue stained SDS-PAGE gel of the purified NuG2-PADC-NuG2. Lane 1: NuG2-PADC-NuG2 (34 kDa). Lane 2: pre-stained protein molecular weight marker. 2.5.2 Preparation of dsDNA Handles and dsDNA-Protein Chimera. The dsDNA handles were prepared largely according to the protocol described by Cecconi et al.219 and Zhang et al.220 Two dsDNA handles of 802 and 558 bp were generated via regular PCR amplification. The template was a pGEMEC-1 plasmid, the reverse primers were the 5’-amine group modified and the forward primers were 5’-biotin (or 5’-digoxigenin) modified, respectively, which were purchased from Integrated DNA Technologies (IDT Inc, San Jose, CA). The sequences of primers for preparing dsDNA handles are shown in Appendix. After the PCR amplification, the PCR products were purified by using a QIAquick PCR purification kit (QIAGEN, Germantown, MD) to remove extra primers. The length of the DNA handles is 802 bp (biotin-DNA-amine) and 558 bp (digoxigenin-DNA-amine), which corresponds to a contour length of 273 nm and 190 nm, respectively. Then dsDNA handles were allowed to react with 4-(N-maleimidomethyl) cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC, SIGMA-56 Aldrich, St. Louis, MO) overnight, which replaced the 5’-amine group by the maleimide group. The freshly expressed proteins were reduced with 1 mM tris(2-carboxyethyl) phosphine (TCEP, SIGMA-ALDRICH, St. Louis, MO) for 1 hour and purified by Zeba desalting columns (7kMW, Thermo Fisher Scientific, Waltham, MA) to remove the extra TCEP. The reduced proteins were diluted to ~3 μM by tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (20 mM Tris, 150 mM NaCl, pH 7.4) and 1 μL of the diluted protein was allowed to react with 1 μL of 3 μM prepared DNA handles at room temperature overnight. The DNA-protein chimera was diluted to ~10 nM and stored at -80 °C. 2.5.3 Single-Molecule OT Experiments. Here we used a MiniTweezers setup developed by Dr. Steven B. Smith for our studies.120 Figure 2.7 shows the experimental setup. Two diode lasers (SDL 5431, 835 nm, 200 mW) are used to generate two counter propagating laser beams. At the end of each laser diode, there is an optical fiber that transmits the light. Two piezoelectric actuators are used to change and control the orientation and position of each beam during the experiment. Objectives are Nikon 60× plan-Apo-water NA 1.2 with correction collars. Position sensitive detectors are used to measure the position of the beam. 57 Figure 2.7 Schematics of the optical setup used in the MiniTweezers. Image adapted with permission from Springer, Copyright © 2020.221 The single-molecule OT experiment was conducted on this MiniTweezers setup.120 1 μL of the streptavidin-coated beads (1% w/v 1 μm, Spherotech Inc., Lake Forest, IL) was diluted to about 3 mL Tris-HCl buffer (20 mM Tris, 150 mM NaCl, pH 7.4) and injected into a liquid chamber. A single streptavidin-coated bead was captured by the laser beam and moved onto the tip of a micropipette in the chamber. The bead could be sucked firmly by applying a vacuum through the micropipette. Then 1.5 μL of the diluted dsDNA-protein chimera sample was added to 4 μL of antidigoxigenin-coated beads (0.5% w/v, 2 μm, Spherotech Inc., Lake Forest, IL) for 30 min to initiate the specific antigen-antibody interaction. The antidigoxigenin-coated beads carrying the dsDNA-protein chimera were also diluted to 3 mL Tris-HCl buffer (20 mM Tris, 150 mM NaCl, pH 7.4) and subsequently injected and captured by the light trap. By allowing the antidigoxigenin-coated bead to be in contact with the streptavidin-coated bead held by the micropipette, the interaction between biotin and streptavidin will enable the formation of a 58 molecular tether between the two beads and enable the stretching of the dsDNA-protein-dsDNA construct (Figure 2.1B). 2.5.4 Measuring Force-Extension Relationship of Protein Unfolding Stretching the protein-DNA chimera resulted in force-distance curves, in which the distance contains the contribution of the extension of the protein-DNA construct as well as the compliance of the optical trap. The MiniTweezers setup is a nonlinear optical trap, and its stiffness varies with the stretching force.120 Therefore, force-distance curves measured using the MiniTweezers setup cannot be directly converted into force-extension curves of the protein-DNA construct itself. As such, the force-distance curves cannot be directly fitted to the WLC model of polymer elasticity to measure the ΔLc of protein unfolding. To describe the unfolding of protein alone, a strategy was used to acquire the force-extension relationship of protein from the force-distance curves. For a given unfolding event with a contour length increment of ΔLc in the force-distance curve, the length increase at a given force F gives the extension Δx of the unfolded polypeptide chain with a contour length of ΔLc. By fitting the measured force-extension relationship to the WLC model, we can measure the persistence length and ΔLc of the unfolded polypeptide chain being released from the unfolding.79, 192, 220 The relationship between F and Δx is shown as following: 𝐹(𝛥𝑥) =𝑘𝐵𝑇𝑝[14(1 −𝛥𝑥𝛥𝐿𝐶)−2−14+𝛥𝑥𝛥𝐿𝐶] (2.1) where kB is the Boltzmann constant, T is the absolute temperature and p is the persistence length of protein. 59 2.5.5 Calculating the Unfolding and Folding Kinetics. A direct and model free calculation method proposed by Oesterhelt et al.140 was used to measure the force-dependent unfolding (or folding) rate constants. The force-distance curves, generated by stretching (or relaxing) the target protein at a constant velocity, were divided into small time windows (Δt) and the force can be regarded as constant within the time window. The unfolding and folding rate constant at force F can be calculated as α(F)=N(F)/M(F)×Δt and β(F)=N(F)/M(F)×Δt, where α(F) (or β(F)) is the unfolding (or folding) rate constant under the force F, N(F) is the total number of unfolding events observed at force F and M(F) is the total number of time windows at force F. The force-dependency of unfolding (or folding) rate constants was subsequently fitted to the Bell-Evans model, to extract the intrinsic protein unfolding (or folding) rate constant at zero force.74-75 The Bell-Evans model can be described as α(F) = α0 exp(FΔxu/kBT) and β(F) = β0 exp(-FΔxf/kBT), where kB is the Boltzmann constant, T is the temperature in Kelvin, α0 (or β0) is the intrinsic unfolding (or folding) rate constant at 0 pN and Δxu (or Δxf) is the unfolding (or folding) distance. 2.5.6 SMD Simulations. SMD simulations were carried out to stretch PADC from the N- and C-termini using constant-velocity mode. The coordinate file for protein PADC was obtained from the PDB (PDB: 2QQD), and a protein structure file (psf) was created using VMD.28 The protein was solvated using the Generalized Born (GB) implicit solvent model,222 and the ensemble is NVT.223 The simulation program is NAMD,224 and CHARMM force field topology parameters were used for simulation.225 The system was energy minimized and then equilibrated for 1 ns. The stretching velocity was set 60 at 5 and 10 m/s in the constant-velocity mode. In total, 17 trajectories were generated. VMD and IGOR PRO were used to perform the data analysis. 61 Chapter 3: Mechanically Tightening, Untying and Retying a Protein Trefoil Knot by Single-Molecule Force Spectroscopy 3.1 Synopsis A knotted conformation is one of the most surprising topological features found in proteins, and understanding the folding mechanism of such knotted proteins remains a challenge. Here, we used OT to investigate the mechanical unfolding and folding behavior of the knotted protein TrmD. We found that when stretched from its N- and C-termini, TrmD can be mechanically unfolded and stretched into a tightened trefoil knot, which is composed of about 17 residues. Stretching of the unfolded TrmD involved a compaction process of the trefoil knot at low forces. The unfolding pathways of the TrmD were bifurcated, involving two-state and three-state pathways. Upon relaxation, the tightened trefoil knot loosened up first, leading to the expansion of the knot, and unfolded TrmD can then fold back to its native state efficiently. By using an engineered truncation TrmD variant, we stretched TrmD along a pulling direction to allow us to mechanically unfold TrmD and untie the trefoil knot. We found that the folding of TrmD from its unfolded polypeptide without the knot is significantly slower. The knotting is the rate-limiting step of the folding of TrmD. Our results highlighted the critical importance of the knot conformation for the folding and the stability of TrmD, offering a new perspective to understand the role of the trefoil knot in the biological function of TrmD. 3.2 Introduction A protein knot is one of the most remarkable features found in proteins over the last two decades, and it adds a layer of topological complexity to the protein folding problem.83, 86-87, 90, 197 62 The knot in a protein is believed to be functionally relevant, as well as provide extra structural stability to the protein.226-227 Understanding the molecular mechanism via which knotted protein overcomes the topological barriers to fold represents a significant challenge.198 Both experimental,170, 193-194, 199 including ensemble and single-molecule measurements, and computational studies94, 198, 203 have started to provide invaluable mechanistic insights into the folding of knotted/slipknotted proteins. It has been shown that such knotted proteins are able to overcome the high topological barrier to knot themselves and fold, although generally very slowly.170, 193, 200-201 MD simulations are of great importance to understanding the molecular mechanism of the folding of these proteins. These studies have revealed the complexity of the folding of knotted proteins and proposed several possible knotting mechanisms, including threading, slipknotting, mouse trapping and folding on ribosomes, to offer important insights into the folding mechanism of such knotted proteins at the molecular level.94, 198, 203 TrmD (PDB: 1P9P) possesses a deep trefoil knot within its protein structure.92, 228-229 TrmD is responsible for the methylation of G37 in tRNA containing the sequence of G36pG37, which is critical to preventing frameshifting in the protein translation process.92 TrmD catalyzes the modification process by using S-adenosyl-L-methionine (AdoMet) as the methyl donor, and it is suggested that the conserved knot structure is key to accommodating the adenosine moiety of AdoMet by the loosening and retightening of the trefoil knot during the methylation process.228 Therefore, understanding the folding mechanism of TrmD as well as the structural dynamics of its knotted structure is not only important for elucidating the general folding mechanism underlying knotted proteins, but also key to understanding the functional role of the trefoil knot in the biological function of TrmD. 63 TrmD is an α/β protein and its 3D structure can be readily divided into two subdomains (Figure 3.1A): the N-terminal α/β assembly domain (residues 1-161, colored in blue) and the C-terminal α helical domain (residues 162-246, colored in red).92 The N-terminal α/β assembly domain is a fold related to the Rossmann fold, with a rather compact globular structure, and the C-terminal α helix domain consists of a long disordered polypeptide coil and four relatively flexible α helixes. TrmD contains a deep trefoil knot in its structure, with residues 81-161 (colored in yellow) serving as the knotting loop, and residues 1-80 (colored in blue) and 162-246 (colored in red) as knot tails. Thus, TrmD serves as an ideal model system for investigating the unfolding/folding mechanism of trefoil-knotted proteins. 64 Figure 3.1 Stretching and relaxing TrmD using OT. (A) The 3D structure of the knotted protein TrmD (PDB: 1P9P). TrmD is composed of two subdomains: the N-terminal Rossmann fold with the motif of alternating β strand-α helix-β strand (residues 1-161, colored in blue and yellow) and the C-terminal α helical domain (residues 162-246, colored in red). The knotting loop is colored in yellow (residues 81-161), and the knot tails are colored in blue and red. Residues 9 and 246, along which TrmD is stretched in the OT experiment, are colored in grey. (B) Schematics of the experimental setup of OT. Protein chimera TrmD9,246-(NuG2)2 is coupled with two DNA handles via thiol-maleimide chemistry. The NuG2 domain serves as a fingerprint for identification of single-molecule stretching events. This DNA-protein chimera is then held by two different polystyrene beads, with streptavidin and anti-digoxigenin modified on their surface, respectively. One bead is held by a glass pipette and the other is trapped by the OT. Stretching or relaxation of the protein chimera can be achieved by moving the laser trap. Amongst the experimental techniques used to study knotted and slipknotted proteins, SMFS is of particular advantage due to its ability to manipulate the knot/slipknot by directly stretching the structure, offering a unique perspective to study the knotting/unknotting process of these proteins.170, 190-192, 199, 230 AFM and OT have been exploited for such studies. Due to the relatively low resolution associated with the measured forces (~10 pN), AFM was mainly used to 65 probe the unfolding/untying mechanism of knotted and slipknotted proteins, and direct observation of their folding was largely inaccessible in these studies.190-191, 199 Due to its superb resolution in force (0.1 pN),170, 219-220 OT has just been utilized to investigate the unfolding/folding mechanism of a 52-knotted protein, ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1), and slipknotted proteins, AFV3-109 and PADC.170, 192, 230 However, the unfolding/folding mechanism of a trefoil knotted protein has rarely been investigated by SMFS studies. Due to the simplicity of the trefoil knot, TrmD serves as an ideal model system for the OT study. Here, we combined OT with protein engineering techniques to investigate the unfolding and folding mechanisms of TrmD. Our results showed that upon stretching from its N- and C-termini, TrmD can be mechanically unfolded and stretched into an unfolded polypeptide chain with a tightened trefoil knot. Both two-state and three-state mechanical unfolding pathways were observed. Upon relaxation, the tightened knot can be loosened by thermal energy and the unfolded polypeptide can fold back to its native state efficiently. The folding of TrmD from such an unfolded state containing a knot is fast and robust. Distinct from a typical two-state folding process, the folding of TrmD from an unstructured polypeptide containing a knot involves a loosening process of the tightened trefoil knot prior to the folding into its native state. In addition, by using a truncation mutant of TrmD, we unfolded TrmD and untied the trefoil knot. Our results showed that the folding of TrmD from such an unknotted and unfolded state was much slower, suggesting that the knotting was the rate-limiting step during the folding of the whole knotted protein. 66 3.3 Results 3.3.1 Stretching TrmD Leads to the Mechanical Unfolding of TrmD and the Tightening of the Trefoil Knot. To investigate the mechanical unfolding of the trefoil knotted protein TrmD using OT, we stretched TrmD from its C-terminus (residue 246) and residue 9 using the TrmD variant Phe9Cys (termed as TrmD9,246) (Figure 3.1A). Residue 9 is more solvent-exposed (than the N-terminal end), so that the coupling efficiency of DNA handles to the protein could be significantly improved. We then engineered the dsDNA-protein chimera, DNA-TrmD9,246-(NuG2)2-DNA, by coupling dsDNA handles to the construct TrmD9,246-(NuG2)2 via thiol-maleimide coupling chemistry.192, 206, 220, 230 In this DNA-protein construct, the well-characterized NuG2 domains serve as fingerprints for identifying single-molecule stretching events.206, 230 Stretching TrmD9,246-(NuG2)2 resulted in force-distance curves with the characteristic sawtooth-like appearance (Figure 3.2A, inset). Two unfolding events occurred at higher forces (typically higher than 30 pN) with a ΔLc of 18 nm (colored in black), corresponding to the mechanical unfolding of the fingerprint NuG2 domains. Thus, the third unfolding event at ~10 pN can be readily attributed to the unfolding of TrmD (colored in red). Since TrmD unfolds at forces that are significantly lower than those for NuG2, subsequent stretching of TrmD was limited to ~15 pN so that only TrmD was unfolded and NuG2 domains remained folded in the OT experiments (Figure 3.2A and B). 67 Figure 3.2 Stretching TrmD9,246 to a tightened trefoil knot involves two parallel pathways. (A) Representative force-distance curves showing two-state unfolding of TrmD9,246. The pulling speed was 50 nm/s. Inset: representative force-distance curves showing the unfolding of full-length TrmD9,246-(NuG2)2. The mechanical unfolding events of the fingerprint domains NuG2 are colored in black and indicated with circles, and the unfolding events of TrmD9,246 are indicated with squares. (B) Representative force-distance curves of TrmD9,246 via the three-state unfolding pathway. The pulling speed was 50 nm/s. The zoomed-in view showed that the protein unfolds via an intermediate state. The protein unfolding from N → I and I →U is colored in blue and green, respectively. The dashed lines in (A) and (B) are pseudo WLC fits to the force-distance curves, generated by our self-developed Igor Pro program. The fits were primarily used for identification of various states (folded, intermediate and unfolded state) of protein in the force-distance curves, and the fitting parameters were not physically meaningful. (C) Force-extension relationships of the unfolding of TrmD9,246. Extension is the length increase at a given force upon unfolding. Error bars correspond to the standard deviation of the data measured from different molecules. Dashed lines are the WLC fits to the force-extension data measured for N → I (blue), I → U (green) and N → U (red), respectively. The WLC fits yielded a persistence length of 0.8 nm and a ΔLc1 of 9.8 ± 0.4 nm, ΔLc2 of 29 ± 1 nm, and ΔLc of 37 ± 2 nm, respectively. (D) Unfolding force histograms of TrmD9,246 at a pulling speed of 50 nm/s. Inset: force-dependency of unfolding rate constants measured by the model-free method proposed by Oesterhelt et al. Fitting to the Bell-Evans model yielded the intrinsic unfolding rate constant α0 and the unfolding distance Δxu, as summarized in Table 3.1. 68 Table 3.1 Average unfolding and folding forces and kinetic parameters for TrmD9,246 and TrmD Δhelix45,128 Fu (pN) Ff (pN) α0 (s-1) Δxu (nm) β0 (s-1) Δxf (nm) ΔLc (nm) TrmD9,246 N-U 8 ± 2 (n=371) 4.5 ± 0.7 (n=543) (3.8 ± 0.5) × 10-3 2.9 ± 0.3 (3.3 ± 0.3) × 102 4.4 ± 0.3 37 ± 2 N-I 10 ± 1 (n=172) N/D (3.6 ± 0.6) × 10-3 2.9 ± 0.4 N/D# N/D 9.8 ± 0.4 I-U 9 ± 1 (n=172) N/D (3.6 ± 0.3) × 101 0.5 ± 0.1 N/D N/D 29 ± 1 TrmD Δhelix45,128 N-U 8 ± 2 (n=142) 4.8 ± 0.6 (n=142) (3 ± 2) × 10-1 0.7 ± 0.4 (2.5 ± 0.4) × 103 5 ± 1 25.6 ± 0.7 # N/D: not determined The mechanical unfolding of TrmD9,246 was observed to follow bifurcated pathways, involving both two-state and three-state pathways. Figure 3.2A shows typical force-distance curves of TrmD following the two-state unfolding pathway. Fitting the force-extension relationships to the WLC model of polymer elasticity gave a contour length increment ΔLc of 37 ± 2 nm upon the unfolding of TrmD (Figure 3.2C). TrmD is 246 amino acid residues long, but only 238 residues were involved in the pulling experiments of TrmD9,246. The complete unfolding of TrmD9,246 would lead to a ΔLc of ~80 nm (238 aa × 0.36 nm/aa – 5.4 nm = 80.3 nm, where 5.4 nm is the distance between residues 9 and 246) if there was no knotted conformation. It is evident that the calculated ΔLc is significantly larger than the experimentally measured one, suggesting that part of TrmD9,246 may have unfolded at low forces, which are below the detection limit of the MiniTweezers. It is of note that TrmD contains a large C-terminal α helical domain (residues 133-246). The α helical structure is mechanically labile and often unfolds at very low forces, resulting in the missing of a clear mechanical unfolding signature in SMFS experiments.199, 212 We speculated that the unfolding of the labile α helix (including the C-terminal α helical domain and the α helix in the 69 knot core (residues 133-161)) would account for the missing ΔLc in TrmD. To verify this hypothesis, we engineered an α helix truncation variant TrmD Δhelix (residues 9-161), in which residues 162-246 were deleted. Stretching TrmD Δhelix9,161 resulted in a ΔLc of ~37 nm, identical to that of wt TrmD (Figure 3.3), suggesting that indeed the unfolding of the C-terminal α helix occurred at low forces and in a gradual fashion, and did not result in a clear unfolding signature in the force-distance curves. Figure 3.3 Mechanical unfolding and folding of TrmD Δhelix9,161. (A) Representative force-distance curves showed that the unfolding and folding signatures were identical to those of TrmD9,246. Both two-state and three-state unfolding were observed for protein unfolding. Upon relaxation, the tightened trefoil knot followed a quasi-two state pathway to the native state. (B) Force-extension relationships of the unfolding of TrmD Δhelix9,161. The WLC fits measured a persistence length of 0.8 nm and ΔLc1 of 8.7 ± 0.7 nm, ΔLc2 of 30 ± 2 nm and ΔLc of 38 ± 2 nm, respectively. If the α helix in the knot core (residues 133-161) also unfolds at low forces similar to the C-terminal α helical domain, the unfolding of the rest of the folded protein structure (residues 9-132) would yield a ΔLc of 42.7 nm (124 aa × 0.36 nm/aa – 1.9 nm = 42.7 nm, where 1.9 nm is the distance between Cys9 and Ser132). This ΔLc of 42.7 nm is about 6 nm longer than the experimentally measured ΔLc of TrmD9,246. Distinct from the unfolding of NuG2, the unfolding 70 of TrmD9,246 would result in a tightened trefoil knot present within the unstructured polypeptide chain. Thus, the “missing ΔLc” of ~6 nm, corresponding to ~17 amino acid residues, can be readily attributed to the formation of the tightened trefoil knot. This result is in good agreement with the size of the protein trefoil knot measured previously in SMFS experiments,170, 191 and suggested that TrmD can be mechanically stretched into an unfolded conformation with a tightened trefoil knot. TrmD9,246 unfolds via a two-state pathway, with an average unfolding force of ~8 pN at a pulling speed of 50 nm/s (Figure 3.2D and Table 3.1). Using the method proposed by Oesterhelt et al.,140 we measured the unfolding rate constant as a function of the stretching force (Figure 3.2D, inset). Fitting the experimental data to the Bell-Evans model74-75 gave the intrinsic unfolding rate constant α0 of (3.8 ± 0.5) × 10-3 s-1 at zero force and the unfolding distance Δxu of 2.9 ± 0.3 nm. 3.3.2 The Mechanical Unfolding of TrmD Involved a Short-lived Intermediate State. In our OT experiments, about 68% of TrmD9,246 followed an apparent two-state unfolding pathway. A small portion (~32%) of TrmD9,246 was observed to unfold via a three-state pathway, involving a short-lived unfolding intermediate state (Figure 3.2B). The WLC fits to the extensions at different forces yielded a ΔLc1 of 9.8 ± 0.4 nm and a ΔLc2 of 29 ± 1 nm, respectively (N → I and I → U, Figure 3.2C). The sum of ΔLc1 and ΔLc2 equals 38.7 nm, in good agreement with the ΔLc yielded from two-state unfolding pathway. These results suggested that the mechanical unfolding pathways of TrmD were bifurcated, and the three-state pathway involved a well-defined intermediate state during the mechanical unfolding pathway. In the three-state pathway, the first unfolding event (from the native to the intermediate state) occurred at an unfolding force of 10 ± 1 pN. And the second unfolding event (from the intermediate state to the unfolded state) occurred at 9 ± 1 pN (Figure 3.2D and Table 3.1). It is of 71 note that the unfolding intermediate state was short-lived in the range of force we measured (7-10 pN), with an average dwell-time of ~0.02 s. Fitting the force-dependency of the unfolding rate of the three-state unfolding to the Bell-Evans model yielded the intrinsic unfolding rate constants α0 of (3.6 ± 0.6) × 10-3 s-1 (N → I) and (3.6 ± 0.3) × 101 s-1 (I → U) (Figure 3.2D, inset and Table 3.1), respectively. The unfolding rate constant for I → U was four orders of magnitude larger than that for N → I, suggesting that the intermediate state was rather mechanically unstable and can be unfolded rapidly once the intermediate state is formed. It is of note that the unfolding kinetics parameters for N → I are similar to those of N → U. This similarity together with the short dwell-time of the intermediate state gives rise to the possibility that the observed two-state pathway is just a special case of the three-state pathway, in which, due to the insufficient time resolution, the intermediate was not resolved. Nevertheless, it is important to point out that the observed unfolding intermediate state and the bifurcation of the unfolding pathways are not associated with the trefoil knot, but a property of the folded protein structure itself. 3.3.3 The Compaction of the Trefoil Knot. It is of interest to note that when the mechanical unfolding of TrmD9,246 occurred at ~9 pN, the force-distance curve of the unfolded TrmD largely followed the typical WLC behavior of an unfolded polypeptide chain (Figure 3.2A, curves 1 and 2). However, if the unfolding occurred at lower forces (<6 pN) (Figure 3.2A, curve 3, and Figure 3.4), the force-distance curve of the unfolded TrmD showed a clear deviation from the WLC behavior of an unfolded polypeptide chain (grey dashed line). This behavior suggests that at lower forces, TrmD9,246 first unfolded into a state that is with a shorter Lc, and further stretching caused the lengthening of the unfolded polypeptide 72 chain until its length is the same as that of the unfolded TrmD9,246 with a tightened knot (with the size of 6 nm). This behavior can be explained by the compaction of the trefoil knot by the stretching force as schematically shown in Figure 3.4. At forces higher than 7 pN, the force-distance curve returned to the normal WLC behavior of an unfolded polypeptide chain, suggesting that the trefoil knot had been stretched to a tightened knot at a force of ~7 pN. Further compaction/tightening would require higher forces. Similar compaction behavior was observed for a 52 knot in the pulling experiments of UCH-L1.170 Figure 3.4 Representative force-distance curves showed the mechanical unfolding of TrmD9,246 at low forces. A clear deviation from a typical WLC behavior of an unfolded polypeptide chain can be observed at the early stage. Further stretching leads to the polypeptide lengthening until the force-distance curve returns to follow the WLC behavior. This polypeptide lengthening process corresponds to the compaction and tightening of the knot structure within the protein, which is shown in the schematics on the top. 73 3.3.4 The Unfolded and Knotted TrmD Can Refold Back to Its Native State Upon Relaxation. After its mechanical unfolding, TrmD9,246 was converted into an unfolded polypeptide with a tightened trefoil knot. To investigate whether the unfolded TrmD with a tightened trefoil knot can refold back to its native state, we relaxed the unfolded polypeptide to allow protein refolding. As shown in Figure 3.5A, during the relaxation process (curves colored in black), the protein was observed to refold, characterized by a force jump at ~4 pN and a length shortening to the original length of TrmD9,246. Subsequent stretching led to an identical signature of the unfolding of the knotted protein TrmD9,246, suggesting that the relaxation had resulted in a successful refolding of the native conformation of TrmD9,246. 74 Figure 3.5 Unfolded and tightened TrmD9,246 can fold back to its native state. (A) Representative stretching-relaxation curves of TrmD9,246 at a pulling speed of 50 nm/s. Consecutive stretching and relaxing curves revealed that the tightened protein was able to fold back to the native state. During relaxation, the force-distance relationship of TrmD deviated from the typical behavior of a DNA-protein chimera starting from ~6 pN (as indicated by the dashed pseudo WLC fits). The protein eventually folded back into its native state via a quasi-two-state mechanism at ~4 pN. (B) The force histogram of the “quasi-two-state” folding of TrmD9,246. Inset: force-dependency of the folding rate constants measured using the Oesterhelt method. The dashed line is the fitting using Bell-Evans model. Fitting parameters β0 and Δxf are shown in Table 3.1. However, the folding behavior of TrmD9,246 is distinct from that of a typical two-state folding event. During the relaxation and refolding process of a typical two-state folder, such as NuG2 (Figure 3.6), the unfolded polypeptide chain followed the typical WLC behavior until the refolding event occurs. In contrast, during the relaxation of the unfolded TrmD9,246, the force-75 distance relationship of the unfolded TrmD9,246 polypeptide chain showed a clear deviation from that of WLC when the force was lower than ~7 pN. When the force reached ~4 pN, the unfolded TrmD refolded, as evidenced by a sudden force jump. This behavior seemed to be the reverse of the compaction process of the trefoil knot observed during unfolding (Figure 3.4), suggesting that the global folding of the knotted TrmD9,246 may proceed with the loosening process of the tightened trefoil knot. Figure 3.6 Mechanical unfolding and folding of two NuG2 protein domains at a pulling speed of 50 nm/s. The unfolded NuG2 domains follow the typical WLC behavior of an unfolded polypeptide chain. The “quasi-two-state” folding forces of TrmD9,246 showed a narrow distribution with an average force of ~4.5 pN at a pulling speed of 50 nm/s (Figure 3.5B, and Table 3.1). Moreover, fitting the force-dependent folding rate constants to the Bell-Evans model gave a folding rate constant at zero force of (3.3 ± 0.3) × 102 s-1 (Figure 3.5B, inset). These results indicated that the folding of TrmD from its unfolded state with the pre-formed knot was fast and robust. 76 3.3.5 Untying and Tying the Knotted Structure of TrmD Using OT Having characterized the mechanical unfolding of TrmD9,246 and the folding of unfolded and knotted TrmD9,246, we then investigated the possibility of unfolding TrmD and untying its trefoil knot. The trefoil knot in TrmD is very deep, and even removing 79 residues from either N- or C-termini will not destroy the trefoil knot. This deep knot is difficult to untie in a SMFS experiment. In order to untie the trefoil knot in TrmD, we used the helix truncation variant TrmD Δhelix, in which the depth of the trefoil knot is significantly reduced. Residues Arg45 and Asp128 were mutated to cysteines to allow TrmD to be stretched from these two residues (this construct is termed as TrmD Δhelix45,128) (Figure 3.7A). 77 Figure 3.7 Untying and tying the knotting loop of TrmD Δhelix45,128. (A) Schematics of stretching protein TrmD Δhelix45,128. The stretching sites are colored in grey. (B) Representative stretching-relaxation curves of TrmD Δhelix45,128 showed distinct unfolding behaviors of the same molecule. Relaxation of the unfolded protein led to a “refolding” event at ~4 pN. However, it remained unknown whether the protein folded into its native state with the trefoil knot. In the subsequent stretching force-distance curves, either a typical two-state unfolding at a force ~8 pN or a “hump” feature was observed. (C) Histograms of unfolding and “refolding” forces of TrmD Δhelix45,128 at the pulling speed of 50 nm/s. Inset: force-dependency of the unfolding and folding rate constants. Dashed lines are fits of the experimental data to the Bell-Evans model. (D) Relationship of the knotting probability versus waiting time Δt at zero force. The dashed line is the fit of the experimental data to the first order rate law, yielding a knotting rate constant of (4 ± 1) × 10-2 s-1. Stretching TrmD Δhelix45,128 resulted in the typical force-distance curves shown in the Figure 3.7B. The unfolding of TrmD Δhelix45,128 largely followed a two-state unfolding pathway with an average unfolding force of ~8 pN at a pulling speed of 50 nm/s. Fitting the WLC model of 78 polymer elasticity to the extension of TrmD Δhelix45,128 led to a ΔLc of 25.6 ± 0.7 nm upon the unfolding of TrmD Δhelix45,128 (Figure 3.8). When stretched from residues 45 and 128, 84 residues were involved. The complete unfolding of TrmD Δhelix45,128 would result in a ΔLc of 26.9 nm (84 aa × 0.36 nm/aa – 3.3 nm = 26.9 nm, where 3.3 nm is the distance between Cys45 and Cys128), in excellent agreement with the experimentally measured ΔLc. This result strongly indicated that stretching the TrmD Δhelix from residues 45 and 128 resulted in the mechanical unfolding of the TrmD Δhelix and untying of its trefoil knot, providing a unique opportunity to investigate the knotting and refolding process of TrmD. Figure 3.8 Force-extension relationship of the unfolding of TrmD Δhelix45,128. The WLC fits yielded the persistence length of 0.8 nm and ΔLc of 25.6 ± 0.7 nm. TrmD Δhelix45,128 unfolds via a two-state pathway with an average unfolding force of ~8 pN at a pulling speed of 50 nm/s (Figure 3.7C and Table 3.1). Fitting the force-dependent unfolding rate constants to the Bell-Evans model yields the intrinsic unfolding rate constant α0 of (3 ± 2) × 10-1 s-1 (Figure 3.7C, inset). After the TrmD Δhelix45,128 had been unfolded and untied, the unfolded and unknotted polypeptide chain was relaxed to zero force to allow the folding and knotting of TrmD. During the relaxation process, a “refolding” event was often observed at ~4 pN, leading to a “refolded” state that has a similar length to the native TrmD Δhelix45,128 (Figure 3.7B). 79 The average “refolding” force is ~4 pN and the intrinsic “refolding” rate constant was estimated to be (2.5 ± 0.4) × 103 s-1 (Figure 3.7C, inset). However, it was unclear whether the trefoil knot had reformed in this “refolded” state. To check this, we stretched the “refolded” state in the next cycle. In about 20% of the traces, a clear unfolding event occurring at ~8 pN was observed (Figure 3.7B, curves marked by “N”). The unfolding signatures of these events were the same as those of the native TrmD Δhelix45,128 with the trefoil knot, suggesting that in these “refolded” molecules, the trefoil knot had been retied. However, in 80% of the traces, the unfolding occurred at ~4 pN, resulting in a hump-like feature (Figure 3.7B, curves marked by “M”). This unfolding signature is different from that of unfolding of the native TrmD Δhelix45,128, suggesting that in these events, TrmD Δhelix45,128 folded into a misfolded or intermediate state that is mechanically labile, and the trefoil knot likely had not reformed. Therefore, the observed “refolding” at ~4 pN for TrmD Δhelix45,128 did not reflect the true refolding of the native trefoil TrmD. Instead, it is likely that such “refolding” events reflected the folding into an unknotted, misfolded state or intermediate state. The knotted conformation is an important structural feature of TrmD. Since the mechanical stretching of TrmD Δhelix45,128 involved untying the trefoil knot by pulling the C-terminus of the protein threading out of the knot core, the correct folding of TrmD must involve tying the C-terminal knot tail into the knot core. It has been shown that knotting is the rate-limiting step in the folding of some knotted proteins.170, 217 To investigate if knotting is the rate-limiting step for TrmD Δhelix45,128, we used OT to investigate the folding behavior of TrmD by stretching wt TrmD from its residues 45 and 128 (termed as TrmD45,128). To untie a protein knot, the length of the polypeptide chain between the two stretching points in the protein knot structure should be much longer than the length of the knot tail to be 80 pulled out of the knot. For the pulling geometry of TrmD45,128, TrmD can be unfolded but its deep trefoil knot cannot be untied due to the long C-terminal knot tail (residues 162-246). Our OT experiments showed that the unfolding of TrmD45,128 displayed a ΔLc of 27.6 ± 0.7 nm (Figure 3.9B and C), which is the same as that of TrmD Δhelix45,128. But the folding of TrmD45,128 was drastically different from that of TrmD Δhelix45,128. The folding of TrmD45,128 occurred in a two-state fashion at ~6 pN (Figure 3.9D). Consecutive stretching-relaxation cycles showed that TrmD45,128 could always fold correctly back to its native state, and no misfolding was observed. Kinetics analysis revealed a high folding rate constant at zero force (Figure 3.9D, inset), indicating that TrmD45,128 can fold rapidly in the presence of a pre-formed knotted structure. 81 Figure 3.9 Stretching the protein TrmD45,128 does not untie the knotted conformation. (A) Schematics of stretching protein TrmD45,128. The stretching site is colored in grey. (B) Representative force-distance curves of TrmD45,128. Consecutive stretching-relaxation curves revealed that the protein can always fold back to its native state. (C) The WLC fits to the force-extension relationship of the unfolding of TrmD45,128 measured a persistence length of 0.8 nm and ΔLc of 27.6 ± 0.7 nm. (D) Unfolding and refolding force histograms of TrmD45,128 at the pulling speed of 50 nm/s. The inset shows the force-dependency of the unfolding and folding rate constants. Fitting the experimental data to Bell-Evans model yielded the intrinsic unfolding and folding rate constants, α0 = (8 ± 2) × 10-2 s-1 and β0 = (1.0 ± 0.1) × 104 s-1, as well as the unfolding and folding distance, Δxu = 0.7 ± 0.2 nm and Δxf = 4.9 ± 0.6 nm. The comparison of the folding behavior of TrmD45,128 and TrmD Δhelix45,128 clearly indicated the critical importance of the knotting step in the folding of TrmD. To determine the knotting kinetics of TrmD Δhelix45,128, we used a double-pulse protocol in the refolding 82 experiment.231 In the first pulse, the correctly folded and knotted TrmD Δhelix45,128 was stretched until the protein was unfolded and the trefoil knot was untied, and then the unfolded and untied polypeptide chain was allowed to relax to zero force and held for a period of time Δt. In the second pulse, the protein was stretched again. The occurrence of the unfolding signatures of the correctly folded and knotted TrmD Δhelix45,128 would indicate the successful knotting and folding of the protein. As shown in Figure 3.7D, the percentage of successful knotting and folding events of TrmD Δhelix45,128 increased with the increasing of waiting time Δt. Assuming a first-order kinetics, we estimated a rate of knotting of TrmD Δhelix45,128 of (4 ± 1) × 10-2 s-1. This rate constant is considerably smaller than the folding rate of TrmD with a pre-formed knotting loop, suggesting that the protein knotting is indeed the rate-limiting step for the folding of TrmD Δhelix45,128. Considering the depth of the trefoil knot in wt TrmD, it is likely that the knotting step of wt TrmD is significantly slower than that of TrmD Δhelix, raising an interesting question if the folding of TrmD in vivo involves any chaperones. It is also important to note that the folding of the unfolded TrmD45,128 also showed significant difference from the folding behavior of the unfolded TrmD9,246. The force-distance relationship of the unfolded TrmD45,128 largely followed the WLC model, and no loosening process of the tightened trefoil knot was observed. This behavior can be readily understood by the unfolding behavior of TrmD45,128 schematically shown in Figure 3.9A. In this pulling geometry, after the unfolding of TrmD45,128, the stretching force will not untie the trefoil knot (as occurred for TrmD Δhelix45,128) due to the long C-terminal knot tail, or tighten the trefoil knot (as occurred for TrmD9,246). Instead, the stretching force acting on TrmD45,128 will only expand the trefoil knot structure, leading to the absence of a tightened knot conformation in the unfolded TrmD45,128. Thus, 83 the folding of the unfolded TrmD45,128 does not involve the loosening up of the knot structure, but largely reflects the folding of the 3D structure of TrmD. 3.4 Discussion The deep trefoil knot structure is a common and unique feature of RNA methyltransferases (MTases), which catalyze the methylation of the base of ribose moiety of ribosomal RNA (rRNA) or transfer RNA (tRNA).232 TrmD is a conserved MTase in bacteria and responsible for the methylation of tRNA.92, 228-229 It was proposed that the trefoil knot in TrmD is critical for its function, as the trefoil knot is responsible for accommodating the adenosine moiety of the methyl donor of AdoMet in the methylation process by loosening and retightening of the trefoil knot.228 Thus, correct folding of TrmD into its knotted structure is crucial. Using single-molecule OT, we have investigated the mechanical unfolding, tightening and untying of the trefoil knot, as well as refolding of TrmD starting from its unfolded polypeptide chain with or without a pre-formed knot structure. Our results provided some new insights into the unfolding and folding mechanisms of TrmD. After the mechanical unfolding of TrmD, we observed a mechanical compaction process of the trefoil knot at low forces (<6 pN), which led to the lengthening of the contour length of the unfolded polypeptide chain and tightening of the trefoil knot. To the best of our knowledge, this is the first time that such a compaction process is observed in a simple trefoil knot. The fact that the compaction process was only observed at low forces suggested that the compaction process may have completed in the unfolded polypeptide chain when TrmD was unfolded at higher forces, as shown in curves (1) and (2) in Figure 3.2A. Moreover, after the compaction process had completed, the length of the unfolded polypeptide chain remained largely unchanged upon further stretching, implying that the knot size remained largely constant. And further tightening and even 84 “jamming” of the trefoil knot (shrinking the knot size) will require a much higher force than what can be applied using OT (typically <100 pN). Thus, the stretching of the trefoil knot in the OT experiments most likely only resulted in a limited further tightening. When the force is lower than ~6 pN during the relaxation process of TrmD9,246, the thermal energy is sufficient to loosen up the knot tightened by a force at least up to 50 pN and the knot can then expand (Figure 3.10). The observed compaction and loosening of the trefoil knot at low forces revealed the dynamic nature of the trefoil knot structure in TrmD. It is worth noting that this compaction and loosening of the trefoil knot occurs when TrmD is unfolded. Further experiments to demonstrate that similar processes can occur in the folded TrmD will be critical for a deeper understanding of the functional mechanism of TrmD, as crystallography studies suggested that loosening and tightening of the knot are critical for accommodating the adenosine moiety of the methyl donor of AdoMet in the methylation process by TrmD.228 85 Figure 3.10 High tension does not inhibit the folding of TrmD9,246. After TrmD9,246 and NuG2 were unfolded (in red), the protein was stretched to a high force (~50 pN) and held at this force for 1 min. During the subsequent relaxation, TrmD9,246 was observed to fold into its native state successfully. The inset shows the experimental protocol. It is also of note that such a compaction and loosening process of the trefoil knot was not observed in a trefoil knot prepared from UCH-L1 in a previous OT study.170 Whether such a compaction and loosening process is related to the protein sequence or residual secondary structure remains to be elucidated. Our results showed that the folding of TrmD with a pre-formed knot is fast and robust, and the knotting is the rate-limiting step for the overall folding of TrmD. These findings complement the previous results obtained on knotted proteins (52 knotted UCH-L1 and trefoil-knotted methyltransferases Yibk and YbeA),170, 217 and raise the question if this feature is common among all knotted proteins. It is important to point out that, due to the pulling geometry in TrmD45,128, the unknotting can only occur by threading the C-terminal end out of the knotting core in our OT experiments. In comparison, the depth of the trefoil knot in wt TrmD prevented us from preparing an untied wt 86 TrmD, and thus it is not possible to directly study the knotting and folding process of wt TrmD using OT. Given the depth of the trefoil knot in wt TrmD, it is most likely that the folding of TrmD in vivo is different from what we observed in TrmD45,128. More studies, likely involving chaperones or folding on ribosomes, will be needed to elucidate the folding mechanism of wt TrmD in vivo. 3.5 Conclusions By combining OT and protein engineering techniques, we investigated the mechanical unfolding and folding behaviors of the knotted protein TrmD. Our results showed that TrmD can be mechanically unfolded along a direction defined by two specific amino acid residues. Depending on the pulling geometry, TrmD can be tightened, untied, or expanded in the unfolded state by the stretching force. After the mechanical unfolding of TrmD, a compaction process of the trefoil knot was observed at low forces. The folding of TrmD strictly depends on the conformation of the unfolded TrmD. If the unfolded TrmD contains a pre-formed knot, the folding of TrmD is fast and robust. If the unfolded TrmD does not contain a pre-formed knot, the knotting would be the rate-limiting step for the protein folding. Our studies provided detailed mechanistic insights into the folding and unfolding of TrmD, and revealed the intricate interplay between the folding and knotting process of TrmD. 3.6 Experimental Section 3.6.1 Protein Engineering. The wt TrmD contains two cysteine residues in its sequence (Cys112 and Cys178). To avoid undesired coupling to the designed DNA handles during the coupling reactions, both Cys112 and Cys178 were mutated to serine. A codon-optimized gene encoding the cysteine-free TrmD with desired restriction sites (5’ BamHI and 3’ BglII and KpnI) was custom synthesized (GeneScript). To avoid the inefficient coupling between the DNA handles and the first amino 87 residue of wt TrmD, which was partially buried in the N-terminal subdomain, the 9th amino acid residue phenylalanine was mutated to cysteine (Phe9Cys) using megaprimer approach of site-directed mutagenesis.233 Following our well-established iterative molecular biology strategy,231 we constructed the gene of TrmD9,246-(NuG2)2. The gene TrmD9,246-(NuG2)2 was then subcloned into a modified expression vector pQE80L-Cys, which carried a cassette allowing us to add one cysteine residue only at the C-terminus of the target protein, to build pQE80L/ TrmD9,246-(NuG2)2-Cys. The gene encoding TrmD45,128 was constructed by using megaprimer approach. TrmD Δhelix9,161 and TrmD Δhelix45,128 was obtained by a further regular PCR amplification to remove the C-terminal α helical domain (residues 162-246). TrmD Δhelix45,128 and TrmD45,128 were subcloned into the pQE80L vector for protein expression. All of the constructed genes were confirmed by DNA sequencing. The protein sequences of TrmD9,246, TrmD45,128, TrmD Δhelix9,161, and TrmD Δhelix45,128 are shown in Appendix. Protein expression and purification were performed using the same procedure as described in chapter 2 (2.5.1). SDS-PAGE was performed to confirm the molecular weight of the engineered proteins (Figure 3.11). 88 Figure 3.11 Coomassie blue stained SDS-PAGE gel of different constructs of TrmD. Lane 1, 3 and 7: pre-stained protein molecular weight marker. Lane 2: TrmD9,246-(NuG2)2 (42 kDa). Lane 4: TrmD Δhelix9,161-(NuG2)2 (32 kDa). Lane 5: TrmD45,128 (29 kDa). Lane 6: TrmD Δhelix45,128 (20 kDa). 3.6.2 Preparation of DNA Handles and DNA-protein Chimera. DNA handles and DNA-protein chimera were prepared using the same procedure as described in chapter 2 (2.5.2). 3.6.3 Single-Molecule OT Experiments. The single-molecule OT experiments (Figure 3.1B) were carried out in the same way as described in chapter 2 (2.5.3). 3.6.4 Measuring Force-Extension Relationship of Protein Unfolding The force-extension relationship of protein unfolding was measured in the same way as described in chapter 2 (2.5.4). 3.6.5 Calculating the Unfolding and Folding Kinetics. For two-state unfolding (or folding) process of protein, the force-dependent unfolding (or folding) rate constants were calculated as described in chapter 2 (2.5.5). The three-state unfolding of TrmD9,246 involved the formation of a short-lived intermediate state. The force-dependent 89 unfolding rate constants associated with the unfolding of intermediate state was measured by fitting the dwell-time distribution to eq. (1.4).234 The relationship of probability density vs dwell-time can be described as Pd(t)=α(F)exp(α(F)×t), where α(F) is the unfolding rate constant under the force F, t is the dwell-time of short-lived intermediate state and Pd(t) is the probability density for a specific dwell-time. The Bell-Evans model was subsequently used to extract the intrinsic unfolding (or folding) rate constant at zero force,74-75 as described in chapter 2 (2.5.5). A standard double-pulse protocol231 was applied to measure the knotting kinetics of mutant TrmD Δhelix45,128. The protein was first untied under stretching, and then quickly relaxed to its initial length. After a time delay Δt, a second stretching was applied to the molecule to check if the knotting was achieved again within the waiting time Δt. Protein knotting probability was increased as a function of Δt, and can be expressed as P(Δt)=1-exp(β0×Δt), where β0 is the intrinsic protein knotting rate constant at zero force. 90 Chapter 4: Single-Molecule Force Spectroscopy Reveals the Mechanical Design Governing the Efficient Translocation of RTX Block V 4.1 Synopsis The efficient translocation of the bacterial toxin CyaA from the bacterial cytosol to the extracellular environment via the type I secretion system (T1SS) is essential for the toxin to function. To understand the molecular features that are responsible for the efficient translocation of CyaA, here we used OT to investigate the mechanical properties and conformational dynamics of a RTX domain, RTX block V, in CyaA at the single-molecule level. Our results revealed that apo-RTX block V behaves like an ideal random coil. This property allows the T1SS to translocate RTX block V without overcoming any mechanical resistance. In contrast, the folded holo-RTX block V is mechanically stable, and its folding occurs in a vectorial fashion starting from its C-terminus. Moreover, our results showed that the folding of RTX block V generates a stretching force, which can further facilitate the translocation of RTX block V. Our results highlight the important role played by the Ca2+-triggered folding of RTX block V in its translocation process and provide mechanistic insights into the mechanical design that governs the efficient translocation of RTX block V. 4.2 Introduction Pore-forming protein toxins are virulence factors produced by many pathogenic bacteria and can cause a range of severe diseases.235-238 Understanding the mechanism via which pore-forming protein toxins interact with the host cells at the molecular level is of great importance to the development of therapeutics for effectively treating diseases. 91 CyaA, from the pathogenic Gram-negative bacteria Bordetella pertussis, is one of such pore-forming protein toxins and responsible for whooping cough.113, 239-241 CyaA is secreted via the T1SS from the Ca2+-depleted bacteria cytosol, where the concentration of Ca2+ is usually lower than 100 nM,242-243 to a Ca2+-rich extracellular space. Upon binding of Ca2+, CyaA folds into a Ca2+-loaded structure, which can then penetrate through the cytoplasmic membrane and translocate into host cells. Inside host cells, CyaA can quickly ablate the physiological functions of host cells by converting cytosolic ATP to cAMP at an uncontrolled level.112-114 During this whole process, the C-terminal RTX domains of CyaA play a crucial role. Each RTX domain contains tandem repeats of the glycine- and aspartate-rich nonapeptide consensus sequence, GGXGXDXXX (where X represents any amino acid residue), that can fold into a Ca2+-loaded, righted-handed parallel β-roll structure.111 CyaA contains five RTX blocks at the C-terminus, from block I to V. Upon binding of extracellular Ca2+ ions, RTX domains can fold into a functional structure that facilitates the binding of CyaA to CD11b/CD18 integrin of host cells and its subsequent penetration into host cells.112-114 Located at the C-terminus of CyaA, RTX block V is the first part of CyaA to be secreted/translocated via the T1SS into the Ca2+-rich environment. The efficient secretion/translocation of CyaA is imperative for its function. Translocation of CyaA via the T1SS is a mechanically driven process largely powered by the hydrolysis of ATP.244 Like other translocation machinery, the T1SS has a narrow inner channel through which the target protein is secreted.244 T1SS-mediated translocation likely involves the unfolding of target protein and subsequent threading of the unfolded polypeptide chain through the conduit. Thus, it is of critical importance to elucidate the structural and mechanical designs of CyaA that govern its efficient 92 secretion via the T1SS. Here, we employed OT to investigate the mechanical unfolding/folding properties of a model RTX domain of CyaA, RTX block V (residues 1529-1681). The 3D structure of RTX block V (PDB: 5CVW) consists of two subunits: the N-terminal right-handed β-roll structure (colored in yellow, Figure 4.1A) with bound Ca2+ ions, and C-terminal α/β assembly (colored in cyan, Figure 4.1A).111 The β-roll structure contains 9 RTX motif-like nonapeptides (residues 1529-1614) and 7 bound Ca2+ ions. The Ca2+ ions are coordinated between two neighboring turns by 6 oxygen atoms from backbone carbonyls and carboxyl groups from side chains of Asp and Glu by charge-dipole interactions. At the C-terminus, the α/β assembly (residues 1615-1681) forms a C-terminal cap-structure without Ca2+ ions. One Ca2+ is bound between the β-roll structure and the α/β assembly, which helps integrate the whole protein structure.111 Previous studies suggested that the presence of Ca2+ ions is essential for the folding of RTX block V, as RTX block V will remain disordered in the absence of Ca2+.111, 245-249 93 Figure 4.1 Probing the unfolding and folding of RTX block V using OT. (A) The 3D structure of RTX block V protein (PDB: 5CVW). RTX block V is a Ca2+ binding α/β protein. RTX block V is composed of two subdomains: the N-terminal β-roll structure (colored in yellow) with the bound Ca2+ ions (colored in grey), and the C-terminal α/β cap assembly (colored in cyan). (B) Schematics of the experimental OT setup. Protein chimera NuG2-RTX block V-NuG2 is coupled with two DNA handles via thiol-maleimide chemistry to create DNA-protein chimera. This chimera is then held by two different polystyrene beads, whose surface is modified by streptavidin and anti-digoxigenin, respectively. One bead is held by a glass micropipette and the other is held by the optical trap. By moving the laser trap, the protein chimera can be stretched or relaxed. During this process, unfolding and refolding of proteins can be directly monitored. The folding of RTX block V in vivo can only occur during or after the translocation into the Ca2+-rich environment. During this process, the translocated polypeptide chain is “tethered” to the T1SS translocation machinery. To some extent, the folding of RTX block V under this condition is similar to the folding of a nascent polypeptide on the ribosome.215-216 To better mimic the unfolding/folding process of RTX block V in vivo, here we employed OT15, 161, 220, 250 to investigate the unfolding and folding pathways of RTX block V at the single-molecule level. Our results unveiled key mechanical features of RTX block V, in both folded and unfolded states, that 94 ensure the efficient translocation of RTX block V via the T1SS. Our studies set the stage for more detailed investigations of CyaA at the single-molecule level. 4.3 Results 4.3.1 Apo-RTX Block V Behaves as a Random Coil and Is Mechanically Compliant. During the secretion process, RTX block V is translocated from the Ca2+-depleted cytosol of Gram-negative bacteria into the Ca2+-rich extracellular environment. During this translocation process, which is ATP-driven, any folded structure, either stable or transient, has to be unraveled before or during translocation due to the narrow channel of the T1SS machinery. To understand the mechanical design of RTX block V, we first investigated the conformational and mechanical properties of apo-RTX block V, which represents the initial structure of RTX block V in the Ca2+-depleted cytosol. CD experiments indicated that apo-RTX block V is largely unstructured (Figure 4.2). This result is consistent with previous spectroscopic studies that suggested that apo-RTX block V is intrinsically disordered.111, 245-249 Figure 4.2 Far-UV CD spectra shows that RTX block V is unstructured in the absence of Ca2+ (solid curve) and is folded in the presence 10 mM Ca2+ (dashed curve). 95 To determine the mechanical properties of RTX block V, we constructed the protein chimera NuG2-RTX block V-NuG2, where the well-characterized NuG2 domains serve as fingerprints for identifying single-molecule stretching events. The mechanical unfolding of NuG2 is characterized by a ΔLc of ~18 nm,144, 206 and unfolding forces over 30 pN at a pulling speed of 50 nm/s.206 The observation of two unfolding events of NuG2 in a given force-distance curve ensures that the force-distance curve contains the unfolding signature of RTX block V. Figure 4.3 shows two pairs of representative stretching-relaxation force-distance curves of NuG2-apo RTX block V-NuG2, in which only two unfolding and refolding events of NuG2 with ΔLc of 18 nm are present. Evidently, stretching the apo-RTX block V does not generate any mechanical unfolding force peak, and the relaxation of the unfolded RTX block V simply follows entropic recoiling and does not generate any refolding signature either. Given that CD results had suggested that apo-RTX block V is unstructured, the SMFS results further corroborate that the apo-RTX block V is mechanically compliant and behaves like a random coil in the absence of Ca2+. More importantly, the observation that apo-RTX block V behaves like a random coil suggested that apo-RTX block V does not form any transient structures that could lead to any mechanical resistance to stretching during the translocation process. This feature is beneficial to the translocation of RTX block V and can potentially reduce the ATP consumption of the T1SS machinery. 96 Figure 4.3 Representative force-distance curves showing the unfolding/folding of Nug2-apo-RTX block V-NuG2 at a pulling speed of 50 nm/s. Two unfolding (indicated by grey circles) and refolding events (indicated by black circles) of NuG2 domains were observed, but the mechanical unfolding of apo-RTX block V did not generate any mechanical rupture event, suggesting that apo-RTX block V is a random coil and does not display any mechanical resistance. 4.3.2 Holo-RTX block V Is Folded and Mechanically Stable. After RTX block V is translocated into the extracellular milieu rich in Ca2+, it can bind Ca2+ and fold into a β-roll structure.111, 246 To investigate the mechanical properties of holo-RTX block V, we carried out pulling experiments in the presence of 10 mM Ca2+ to mimic the extracellular Ca2+ environment. Figure 4.4 inset shows a representative force-distance curve of a full-length NuG2-RTX block V-NuG2. It is evident that in addition to the two unfolding events of NuG2 with ΔLc of 18 nm at ~30 pN (colored in green), a new unfolding event was observed at ~15 pN, which can be readily attributed to the mechanical unfolding of RTX block V. Since the unfolding of RTX block V occurs at forces significantly lower than those of NuG2, subsequent stretching was limited to ~20 pN so that only RTX block V was unfolded (Figure 4.4A and B). The unfolding events in Figure 4.4A occurred in a two-state fashion. Fitting the length change of RTX block V versus forces to the WLC model of polymer elasticity measures a contour length increment ΔLc of 45.1 ± 0.4 nm for the unfolding of RTX block V (Figure 4.4C). 97 Figure 4.4 Holo-RTX block V is mechanically stable and unfolds in two parallel pathways. (A) Representative force-distance curves of RTX block V showing the two-state unfolding and folding. The pulling speed was 50 nm/s. Inset: representative force-distance curves of the full-length NuG2-RTX block V-NuG2. The unfolding and refolding events of the fingerprint NuG2 domain are colored in green and indicated by the circles, and the unfolding and refolding events of RTX block V are indicated by squares. (B) Representative force-distance curves of RTX block V following the three-state unfolding/folding pathway. The pulling speed was of 50 nm/s. Zoomed-in views showed that the unfolding and refolding of RTX block V involved an intermediate state. In (A) and (B), the dashed lines are pseudo WLC fits to the force-distance curves. (C) Force-extension relationships of the unfolding of RTX block V. The dashed lines are the WLC fits to the experimental data for RTX block V from N→I (green), I→U (blue) and N→U (red), respectively. The WLC fits yielded the persistence length of 0.8 nm and ΔLc1 of 16.6 ± 0.3 nm, ΔLc2 of 29.0 ± 0.5 nm and ΔLc of 45.1 ± 0.4 nm, respectively. (D) Histograms of unfolding and refolding forces of RTX block V at a pulling speed of 50 nm/s. RTX block V is 153 amino acid residues long. Complete unfolding of RTX block V would result in a ΔLc of 50.6 nm (153 aa × 0.36 nm/aa − 4.5 nm = 50.6 nm, where 4.5 nm is the distance 98 between the N- and C-termini), about 5 nm longer than the experimentally measured ΔLc, suggesting that part of the folded RTX block V was unfolded at very low forces and did not generate any mechanical unfolding signature. It is of note that there is an α helix at the C-terminus of the folded RTX block V (residues 1665 to 1681). It is known that α helices are mechanically labile. Its mechanical unfolding typically occurs at very low forces and does not generate any mechanical unfolding signature.212 Indeed, after the unfolding of the C-terminal α helix, the unfolding of the remaining folded structure (residues 1529 to 1664) would give rise to a ΔLc of 45.8 nm (136 aa × 0.36 nm/aa – 3.2 nm = 45.8 nm, where 3.2 nm is the distance between the Gly1529 and Asp1664), in excellent agreement with the experimentally measured ΔLc. To corroborate this assignment, we constructed a truncation variant RTX Δhelix1529,1664, in which the C-terminal α helix is deleted (residues 1665-1681). Stretching RTX Δhelix1529,1664 resulted in force-distance curves similar to those of wt RTX block V (Figure 4.5A), and the WLC fits to the force-dependent length increment yielded ΔLc that is identical to that of wt RTX block V (Figure 4.5B). This result strongly indicated that the mechanical unfolding event observed in the OT experiments corresponds to the mechanical unfolding of the folded β-roll structure, which is followed by the unfolding of the C-terminal α helix at low forces. Moreover, the average unfolding force of RTX block V is around 15 pN at a pulling speed of 50 nm/s, suggesting that the folded RTX block V is mechanically stable under physiological conditions (Figure 4.4D).251-253 99 Figure 4.5 OT experiments showed that the deletion of α helix at the C-terminus does not affect the unfolding and refolding pathways of RTX block V. (A) Representative curves showing unfolding and folding of RTX Δhelix1529,1664 via bifurcated pathways at a pulling speed of 50 nm/s. (B) Force-extension relationships of the unfolding of RTX Δhelix1529,1664 in different unfolding pathways (N→I, I→U and N→U). Dashed lines are the WLC fits to the experimental data, which yield the persistence length of 0.8 nm and ΔLc of 16.4 ± 0.2 nm, 29.1 ± 0.7 nm and 45.5 ± 0.4 nm, respectively. The unfolding events of RTX block V with a ΔLc of ~45 nm occurred in an all-or-none fashion, suggesting that these RTX block V molecules unfolded in a two-state fashion. However, it is of note that only a small portion of RTX block V (~15%) followed the two-state unfolding pathway. Most RTX block V (~85%) were observed to unfold following a three-state pathway involving the formation of an unfolding intermediate state (Figure 4.4B). Fitting the WLC model to the length change at different forces revealed a ΔLc1 of 16.6 ± 0.3 nm and a ΔLc2 of 29.0 ± 0.5 nm (Figure 4.4C, green and blue dashed lines), giving rise to a sum ΔLc of 45.6 nm, in good agreement with the ΔLc measured from the two-state unfolding pathway. Taken together, these results suggested that the unfolding of the folded RTX block V follows two parallel pathways, with ~15% of RTX block V following the two-state unfolding pathway, and the remaining 85% following a three-state pathway (Table 4.1). It is worth noting that due to the limited temporal 100 resolution of the MiniTweezers setup (~1 ms), some short-lived intermediate states might not be resolved in our OT experiments, and the observed two-state pathway was just a special case of the three-state pathway. Nevertheless, in both pathways, the unraveling of the C-terminal α helix occurred at low forces and preceded the main unfolding event. Table 4.1 Average unfolding and folding forces and kinetic parameters for wt RTX block V and RTX Δhelix1529,1664 The average unfolding force of RTX block V is around 14 pN and 13 pN at a pulling speed of 50 nm/s for the two-state and three-state unfolding pathways (Figure 4.4D and Table 4.1), respectively, suggesting that indeed RTX block V is mechanically stable. 4.3.3 Unfolded RTX block V Can Refold Rapidly Against a Stretching Force Following Parallel Pathways. Upon relaxation, a force-jump event at ~5 pN was clearly visible in the relaxation curves of RTX block V, suggesting that the unfolded RTX block V is able to refold in the presence of Ca2+ even against a stretching force of ~5 pN (Figure 4.4A and B). Similar to the mechanical unfolding, the refolding of RTX block V was also observed to follow both two-state and three- Fu (pΝ) Ff (pΝ) α0 (s-1) Δxu (nm) β0 (s-1) Δxf (nm) N-U 14 ± 3 (n=160) 3.7 ± 0.5 (n=821) (7 ± 2) × 10-3 1.6 ± 0.3 (2.8 ± 0.3) × 105 12 ± 1 wt RTX block V N-I 13 ± 3 (n=890) 4.4 ± 0.7 (n=214) (4.6 ± 0.5) × 10-2 1.1 ± 0.1 (2.8 ± 0.1) × 103 3.1 ± 0.2 I-U 12 ± 2 (n=890) 3.8 ± 0.6 (n=214) (7 ± 2) × 10-1 1.6 ± 0.2 (8.2 ± 0.6) × 103 8.4 ± 0.6 N-U 13 ± 3 (n=313) 2.4 ± 0.5 (n=903) (1.5 ± 0.6) × 10-2 1.6 (3.6 ± 0.4) × 103 12 RTXΔhelix1529,1664 N-I 10 ± 2 (n=685) 2.9 ± 0.8 (n=113) (1.0 ± 0.6) × 10-1 1.1 (2.7 ± 0.2) × 103 3.1 I-U 9 ± 2 (n=685) 2.8 ± 0.5 (n=113) 1.4 ± 0.5 1.6 (3.7 ± 0.3) × 102 8.4 101 state pathways. The observed ΔLc1 and ΔLc2 are the same as that for the three-state unfolding, implying that the unfolding and refolding in the three-state pathway likely involve a similar (if not the same) intermediate state. The average refolding force of RTX block V is ~5 pN at a relaxation speed of 50 nm/s (Table 4.1), indicating that in the presence of Ca2+, RTX block V can refold efficiently. Clearly, the mechanical unfolding and refolding pathways of RTX block V are bifurcated. About 85% of RTX block V unfolds following the three-state pathway, while 15% of RTX block V follows a simple two-state process. In the folding process, the partition of the two folding pathways is different: ~80% of RTX block V folds in a two-state fashion while only ~20% of RTX block V folds following the three-state pathway (Table 4.1). 4.3.4 Structure of the Unfolding and Folding Intermediate States. The 3D structure of the folded RTX block V contains two subunits, the N-terminal right-handed β-roll structure with bound Ca2+ and the C-terminal α/β cap assembly.111 The observation of an unfolding/refolding intermediate state in RTX block V suggested the existence of a stable partially folded structure. However, it is unclear from which terminus the unfolding of RTX block V starts and what structure the unfolding and refolding intermediate states show. To address these questions, we constructed two variants: RTX block V-L5 and RTX block V A1615C. We first engineered a loop insertion variant RTX block V-L5, where five amino acid residues (GGGLG) were inserted into the unstructured loop between residue Gly1635 and Thr1636 close to the C-terminus of RTX block V (Figure 4.6A). If the unfolding of RTX block V started from the C-terminus, the five inserted residues would lead to an increase of ΔLc1 by ~1.8 nm for the three-state unfolding as well as ΔLc for the two-state unfolding. Similar to wt RTX block V, upon stretching, RTX block V-L5 exhibited two distinct unfolding pathways, namely two state 102 and three-state. The two-state unfolding showed a ΔLc of 47.1 ± 0.8 nm, showing an about 2 nm increase compared with that of wt RTX block V. However, in the three-state unfolding pathway, ΔLc1 remained as ~16.5 nm, while ΔLc2 increased to 31.3 nm, about 2 nm increase over that of wt RTX block V. This result suggested that upon stretching, the N-terminal part of RTX block V unfolded first, and the C-terminal part of the structure served as a stable intermediate state in the three-state unfolding pathway (Figure 4.6B and C). Figure 4.6 OT experiments revealed the structure of unfolding/folding intermediate state. (A) Schematics of protein RTX block V-L5. The location where additional five amino residues (GGGLG) are inserted is indicated schematically. (B) A representative curve showed unfolding and folding of RTX block V-L5 protein via three-state pathway at a pulling speed of 20 nm/s. (C) The WLC fitting of force-extension relationships of the unfolding of RTX block V-L5 from N→I (green), I→U (blue) and N→U (red), respectively. The WLC fittings yield the persistence length of 0.8 nm and the ΔLc of 16.5 ± 0.2 nm, 31.3 ± 0.5 nm and 47.1 ± 0.8 nm, respectively. (D) Schematics of stretching RTX block V A1615C from its N-terminus and residue A1615C (indicated by the red asterisk). (E) A representative force-distance curve showed the unfolding and folding of RTX block V A1615C via a three-state pathway when it is stretched from its N-terminus and residue 1615. (F) The WLC fitting of force-extension relationships of the unfolding of RTX block V A1615C protein from N→I (green), I→U (blue) and N→U (red), respectively. These fits yield the persistence length of 0.8 nm and the ΔLc of 17.2 ± 0.3 nm, 11.1 ± 0.2 nm and 28.6 ± 0.5 nm, respectively. 103 To further corroborate this unfolding mechanism, we also constructed a point variant RTX block V A1615C. Residue Ala1615 is located in the C-terminal α/β cap assembly structure of RTX block V. Stretching RTX block V from residue 1615 and the N-terminus allows for the N-terminal part of the structure to be mechanically unraveled (Figure 4.6D). If the above unfolding mechanism is correct, we should expect that the ΔLc1 will not be affected when RTX block V is stretched from residue 1615 and the N-terminus. As shown in Figure 4.6E and F, indeed, the mechanical unfolding and folding intermediate states were clearly observed during the unfolding and folding processes, and ΔLc1 remained the same as that of wt RTX block V (with ΔLc1 of ~17 nm), but the ΔLc2 was shortened to ~11 nm. Taken together, these results strongly indicated that the mechanical unfolding initiates from the N-terminus, while the folding starts from the C-terminus. Together with the structural information on RTX block V, our results allowed us to depict a detailed picture of the unfolding and folding pathways of RTX block V. The mechanical unfolding pathways are bifurcated: ~15% of RTX block V unfolds in a two-state fashion without the accumulation of any intermediate state; ~85% of RTX block V unfolds following a three-state pathway involving an unfolding intermediate state. In this pathway, upon stretching, the C-terminal α helix (from Gln1665 to Pro1681, colored in purple, Figure 4.7) is unraveled at low forces in a gradual fashion. Then the N-terminal part of β-roll structure (from Gly1529 to Asp1580, involving five β strands and four Ca2+ ions, 52 aa × 0.36 nm/aa – 1.8 nm = 16.9 nm, colored in red, Figure 4.7) unfolds, followed by the unfolding of the rest structure at the C-terminus (from Asp1580 to Asp1664, involving nine β strands and other four Ca2+ ions, 85 aa × 0.36 nm/aa – 2.3 nm = 28.3 nm, colored in yellow, Figure 4.7). 104 Figure 4.7 Schematics of two-state and three-state unfolding/folding pathways of RTX block V. After the unfolding of α helix (colored in purple), the five β strands at the N-terminus (colored in red) will unfold, followed by the unfolding of the rest protein structure (colored in yellow) in the three-state pathway. Black arrows indicate the unfolding process, grey arrows indicate the folding process. The thickness of the arrows indicates the flux of one particular pathway. In the folding process, ~80% of RTX block V can fold following a two-state pathway to acquire its folded structure in a cooperative fashion. In contrast, ~20% of RTX block V folds in a three-state folding process involving a folding intermediate state. In this pathway, the folding initiates from the C-terminus, and the nine C-terminal β strands fold into a stable intermediate state. Then the rest of the β-roll structure folds accompanied by binding of Ca2+ ions. It is of note that only when the stretching force is relaxed to low forces, the C-terminal α helix can fold and RTX block V is mechanically folded into its native state. This three-state folding mechanism provides a means for RTX block V to fold even if only part of RTX block V is translocated into the Ca2+-rich extracellular environment. 105 4.3.5 Kinetics Analysis of Unfolding and Folding of RTX block V. Having established the mechanical unfolding and folding pathways of RTX block V, we determined the kinetic parameters that characterize the mechanical unfolding and folding of RTX block V. Using the method proposed by Oesterhelt et al.,140 we calculated force-dependent unfolding and folding rate constants of RTX block V at different forces from the OT experiments operated in constant-velocity mode (Figure 4.8). Fitting the force-dependency of the unfolding rate constants to the Bell-Evans model,74-75 we measured the intrinsic unfolding rate constant at zero force (α0) and the unfolding distance (Δxu) for the two unfolding pathways. Similarly, we also determined the intrinsic folding rate constant at zero force (β0) and the folding distance (Δxf) for the two different folding pathways (Table 4.1). Figure 4.8 Force-dependency of the unfolding and refolding rate constants of RTX block V for the pathways of (A) N ↔U, (B) N ↔I and (C) I ↔U. Unfolding is colored in red and refolding in blue. The unfolding and folding rate constants were calculated using the Oesterhelt method and the fitting of dwell-time distribution. Dashed lines correspond to the fits of Bell-Evans model to the experimental data. The kinetic parameters are summarized in Table 4.1. It is important to note that at zero force, RTX block V folds extremely fast in both folding pathways (with β0 of 2.8 × 105 s-1 and 8.2 × 103 s-1 for the two-state and three-state pathway, respectively). Such high folding rate constants suggested that the unfolded RTX block V has a 106 very high folding propensity in the presence of Ca2+, allowing for the newly translocated chain to fold rapidly to gain mechanical resistance to unfolding. 4.3.6 C-terminal α helix Stabilizes the Folded β-roll Structure and Facilitates Protein Folding. Although the unfolding and folding of the C-terminal α helix is largely “invisible” in the OT experiments, the deletion of the α helix has a clear effect on the kinetics for the unfolding and folding reactions of RTX block V. Compared with wt RTX block V, RTX Δhelix1529,1664 displayed a lower unfolding force (12.7 pN versus 14.2 pN for wt RTX block V), and thus an increased unfolding rate constant α0 (Table 4.1 and Figure 4.9). In addition, RTX Δhelix1529,1664 also showed a decreased folding rate constant β0 in both two-state and three-state folding pathways, suggesting that the C-terminal α helix is partially structured at the folding transition state and can speed up the folding reaction. These results suggested that the C-terminal α helix, even though partially folded, is important to facilitate the protein folding and stabilize the folded β-roll structure. However, the origin of this phenomenon remains unclear and requires further detailed investigations. 107 Figure 4.9 Signatures of the mechanical unfolding (red) and refolding (blue) of RTX Δhelix1529,1664. (A, C, E) Unfolding and refolding force histograms for the pathways of (A) N↔U, (C) N↔I and (E) I↔U at pulling speed of 50 nm/s, (B, D, F) Force-dependent unfolding and refolding rates calculated by Oesterhelt method and the fitting of dwell-time distribution. The average unfolding and folding forces and kinetic parameters are summarized in Table 4.1. It is also of note that the deletion of the α helix also led to a change of the partition of the two different unfolding pathways for RTX block V. The percentage of three-state unfolding showed a considerable decrease compared with wt RTX block V (69% versus 85% for three-state unfolding), suggesting that RTX Δhelix1529,1664 unfolds in a more cooperative fashion. 4.3.7 Cation-π-π Interactions Play Important Roles in the Folding of C-terminal Structure. It was proposed that the spatial arrangement of residues of Arg1624, Try1646 and Trp1645 in the hydrophobic core of RTX block V can enable a cation-π-π interaction between the positively 108 charged guanidium group of Arg1624 and the aromatic benzene and indole rings of Try1646 and Trp1645. This interaction plays important roles in the folding of the C-terminal structure.111 To further examine this mechanism, we engineered a double mutant W1645G-Y1646G, where both Trp1645 and Tyr1646 were mutated to glycine to disrupt the proposed cation-π-π interaction. Figure 4.10A shows the representative force-distance curve of RTX block V W1645G-Y1646G. Different from wt RTX block V, the vast majority of this mutant (~95%) unfolded following a two-state unfolding pathway, showing a ΔLc of 46.7 ± 0.9 nm (Figure 4.10B). Only 5% of the molecules were observed to unfold in a three-state fashion. Figure 4.10 Mechanical unfolding and folding of RTX block V W1645G-Y1646G are investigated by OT. (A) Representative curves showing unfolding RTX block V W1645G-Y1646G via two-state process at a pulling speed of 20 nm/s. During relaxation, no clear refolding event was observed. (B) Force-extension relationship of the unfolding of RTX block V W1645G-Y1646G. Dashed line is the WLC fit to the experimental data, yielding a persistence length of 0.8 nm and ΔLc of 46.7 ± 0.9 nm. The double mutation has an even more profound impact on the folding of RTX block V. Different from wt RTX block V, relaxing unfolded RTX block V W1645G-Y1646G did not lead to the observation of a refolding event (Figure 4.10A). In addition, subsequent stretching curves often did not show any unfolding event of RTX block V W1645G-Y1646G if the molecule was 109 stretched immediately after it was relaxed to zero force, suggesting that the double mutation significantly slowed down the folding reaction. Using a double-pulse protocol,231 we estimated that the folding rate of RTX block V W1645G-Y1646G at zero force is ~0.05 s-1, 7 orders of magnitude slower than that of the wt RTX block V (Figure 4.11). This result corroborates that the cation-π-π interaction is essential for the fast folding of RTX block V in the presence of Ca2+. Figure 4.11 Cation-π-π interaction is critical for the rapid folding of RTX block V. (A) Unfolding force histograms of RTX block V W1645G-Y1646G protein for the two-state pathway at pulling speed of 20 nm/s. (B) Force-dependent unfolding rates calculated by Oesterhelt method. Dashed lines correspond to the fitting of Bell-Evans model with α0 of (2.6 ± 0.6) × 10-2 s-1 and Δxu of 1.3 ± 0.3 nm. (C) Relationship of folding probability versus folding time at zero force. Fitting the data to the first order kinetics would generate a folding rate constant β0 of (5.4 ± 0.2) × 10-2 s-1. 4.4 Discussion Efficient translocation of RTX block V through the conduit of the T1SS machinery is the first and most critical step for bacterial toxins to properly perform their function, and the structure of RTX block V must have evolved to be uniquely designed for this important task. Understanding 110 how the mechanical motif of RTX block V ensures its efficient translocation is thus imperative for understanding the virulence of these toxins and helps develop therapeutics for combatting these diseases. The translocation of RTX block V is a mechanically driven process along a confined space of the T1SS machinery, accompanied by the consumption of ATP. Although the complete folding of RTX block V can occur only after the polypeptide chain has translocated into the Ca2+-rich environment, partial folding can occur as soon as part of the polypeptide chain has emerged from the translocation machinery, in a way similar to the cotranslational folding of nascent polypeptide chains on the ribosome.202, 215-216 Compared with ensemble studies, SMFS is a uniquely suitable technique allowing for the characterization of the unfolding/folding of RTX block V along a quasi-1D reaction coordinate, which is similar to that occurring during translocation. The structural/biophysical features of RTX block V revealed in this study may help develop new ways to inhibit the proper folding of CyaA. 4.4.1 Domain Structure of RTX block V Does Not Correspond to the Intermediate State Formed in the Unfolding/Folding Process. From a structural point of view, the structure of RTX block V can be readily divided into two subunits: the N-terminal right-handed β-roll structure with bound Ca2+ ions and the C-terminal α/β cap-assembly.111, 247 This division, however, does not necessarily reflect the intermediate structure formed during the unfolding (or folding) process of RTX block V. Our single-molecule OT results on RTX block V-L5 and RTX block V A1615C clearly showed that the C-terminal α/β assembly does not function as an independent folding unit in the three-state pathway. Instead, the C-terminal α/β assembly and the adjacent β strands fold together as a cooperative unit in the presence of Ca2+, and serve as an on-pathway intermediate state for RTX block V. The rest of the 111 β-roll structure folds subsequently. This pathway differs from the previously proposed one based on ensemble methods, in which the C-terminal α/β assembly was proposed to fold on its own.111 Our results suggested that the packing of additional β strands and the binding of Ca2+ may be critical for the stability of the C-terminal α/β assembly structure. 4.4.2 Mechanical Design of RTX block V Ensures the Efficient Translocation. Translocation of RTX domains is a critical step for bacterial toxins to properly function. Our SMFS results on RTX block V now revealed key and unique mechanical features that help explain why RTX block V is so efficient in accomplishing the translocation step. The T1SS translocation machinery is responsible for translocating RTX domains from a Ca2+ deficient bacterial cytosol to Ca2+-rich environment. The translocation by the T1SS is largely driven by ATP,254-255 and requires the protein polypeptide chain to be threaded through a narrow conduit, which only allows an unfolded polypeptide chain to go through. This structural feature of the T1SS translocation machinery requires that the protein is unfolded prior to its translocation. Thus, any folded structure that is present in the toxin and exhibits mechanical resistance to unfolding would require a higher consumption of ATP and slow down the translocation, making the translocation less efficient. In the absence of Ca2+, RTX block V behaves like an ideal random coil, and stretching such an unstructured polypeptide chain only requires overcoming the entropy of the polypeptide chain. This structural feature of apo-RTX block V can potentially lower the consumption of energy and speed up the translocation. Equally important, additional features of RTX block V also make the translocation efficient when the protein is translocated into the Ca2+-rich environment. The signal sequence for translocation is located at the C-terminus of RTX block V,113 and thus the translocation initiates from the C-terminus. After the translocated RTX block V emerges from the T1SS machinery exit 112 channel in the Ca2+-rich environment, it is essential that the polypeptide can quickly fold into a stable structure. A mechanically stable structure could anchor the translocated polypeptide chain in the extracellular environment and prevent its backsliding. The mechanical features of RTX block V again perfectly fulfill this requirement. The C-terminal α helix sequence could fold as soon as it emerges from the translocation conduit; however, the α helix is mechanically labile and not able to resist backsliding on its own. The polypeptide sequence encompassing residues 1681-1580 holds the key. This sequence can refold rapidly (with the intrinsic folding rate constant of ~8000 s-1) in the presence of Ca2+ to acquire a stable partially folded structure. This folding intermediate state can serve as a mechanically stable roadblock to prevent the backsliding and provide a stable anchoring point for the translocation to proceed continuously. After the complete folding of RTX block V, the mechanical stability is further enhanced, consolidating the translocated protein fragment. It is important to point out that the binding of Ca2+ to the structure of RTX block V is critical for mechanical stability. The β-roll structure on its own is not mechanically stable, and the mechanical stability of RTX block V relies on the binding of the eight Ca2+ ions in the structure, which “staple” the β-roll structure together and provide much needed mechanical resistance to the mechanical unfolding. This interesting design also raises an interesting question how this β-roll toxin evolved to use Ca2+ to provide additional mechanical stability. Moreover, protein folding can also provide a further driving force to facilitate translocation. The high folding rate constant of the folding intermediate state at the C-terminus suggested that the folding could occur even when a short stretch of the protein sequence (residues 1681-1580) has exited the translocation conduit. The folding of the C-terminal intermediate state leads to the shortening of the polypeptide chain by ~13 nm against a stretching force of ~5 pN. In the case of 113 translocation, the shortening can lead to an increase of the force experienced by the polypeptide chain in the T1SS conduit. This folding-generated force will not only prevent backsliding of the translocated sequences, but also actively facilitate the translocation by pulling on the polypeptide chain that remained in the translocation channel. This folding-driven power generation shares some similarity with the power-stroke during muscle contraction.256-257 It is interesting to note that a similar folding-driven power generation was observed to play an important role in the folding of nascent chain on ribosome,258 as well as in work generation from protein folding-based soft actuators.259 Since CyaA has multiple RTX domains in the structure, the folding of each RTX domain is expected to further accelerate the translocation, and make the translocation of the whole CyaA more efficient. 4.4.3 Translocation Mechanism of RTX block V. Taking all these unique features together, we propose the following four-step mechanism as an important supplementary mechanism to the ATP-driven, T1SS-mediated translocation mechanism for RTX block V (Figure 4.12). 114 Figure 4.12 Schematics of proposed translocation mechanism of RTX block V. Step 1, driven by the hydrolysis of ATP, the T1SS machinery threads the unfolded RTX block V into the narrow channel of the T1SS. The step lasts until the major part of the C-terminal sequence emerges in the Ca2+ rich environment; Step 2, folding of the C-terminal sequence into the stable intermediate state. This process leads to the shortening of the polypeptide chain and the generation of additional stretching force to the polypeptide chain remaining in the T1SS channel; Step 3, threading the N-terminal sequence (colored in red) until most of it emerges from the T1SS conduit; Step 4, the complete folding of RTX block V, leading to another force generating event. This process leads to the complete translocation of one RTX domain. Repeating step 1-4 might lead to the sequential and vectorial translocation of the other RTX domains. The stretching force experienced by the unfolded polypeptide chain is indicated by the arrow, the length of the arrow indicates the amplitude of the force. Step1. Driven by the ATP, the T1SS translocation machinery threads the disordered sequence into the narrow channel without the need to overcome mechanical resistance of unraveling any folded structures in RTX block V. This process lasts until the emergence of most of the C-terminal sequence of RTX block V (residues 1681-1580) in the Ca2+-rich environment from the translocation conduit. Step 2. Binding of Ca2+ ions to RTX block V allows the translocated sequence to fold rapidly into a stable folding intermediate state, which results in a shortening of the polypeptide chain and the generation of an additional stretching force acting on the polypeptide chain that 115 remains in the translocation machinery channel, resulting in a further translocation. As such, the folding of the intermediate state prevents the backsliding of RTX block V and facilitates the translocation of the polypeptide remaining in the T1SS conduit. Step 3. The N-terminal sequence of RTX block V is threaded via the T1SS to enter the Ca2+-rich environment. Step 4. Folding of the complete RTX block V finishes, leading to another force-generating event and further facilitating the translocation of other RTX domains. Repeating step 1 to 4 leads to the translocation of the remaining RTX domains vectorially and sequentially from the bacterial cytosol to the extracellular milieu. Since RTX from block I to IV of CyaA share a similar consensus sequence, these domains will likely follow a similar folding and translocation mechanism. The mechanism we proposed here is largely in line with the ratchet mechanism proposed previously based on solution studies,111 but adds new important mechanistic information on the translocation process of RTX block V. The mechanism shown in Figure 4.12 provides a detailed picture of the ratchet mechanism in terms of the mechanical stability of RTX block V. More importantly, our mechanism revealed that the folding of RTX block V can generate a stretching force, which can in turn provide an additional driving force to actively accelerate the translocation of the polypeptide chain remaining in the T1SS machinery. However, the amplitude of the folding-generated stretching force remains to be directly determined. 4.5 Conclusions Using single-molecule OT, we have investigated the mechanical unfolding and folding behaviors of RTX block V from the bacterial toxin CyaA. Our results showed that the apo-RTX block V behaves as a random coil and does not show any mechanical resistance to stretching. Upon 116 binding of Ca2+ ions, RTX block V folds into a mechanically stable structure. The unfolding and folding of the holo-RTX block V followed bifurcated pathways, both involving two-state and three-state pathways. The folding of RTX block V initiates from the C-terminus, which can fold into a stable intermediate state to facilitate the folding of the rest of RTX block V. Our results revealed key features in the mechanical design of RTX block V that make it possible for RTX block V to be efficiently translocated from the bacterial cytosol. 4.6 Experimental Section 4.6.1 Protein Engineering. The plasmid encoding RTX block V with the restriction sites (5’ BamHI and 3’ BglII and KpnI) was a generous gift from Dr. Shanshan Lyu from Beijing University of Chemical Technology. Following a well-established iterative molecular biology strategy,231 we constructed the gene of NuG2-wt RTX block V-NuG2. The NuG2-wt RTX block V-NuG2 gene was then subcloned into the expressing vector pQE80L-CC, which was constructed as described,192, 206 to build pQE80L/Cys-NuG2-RTX block V-NuG2-Cys. The gene encoding RTX Δhelix1529,1664 was constructed using polymerase chain reaction. The variants RTX block V W1645G-Y1646G and RTX block V A1615C were obtained using megaprimer approach of site-directed mutagenesis. In the mutant RTX block V-L5, 5 additional amino residues GGGLG were inserted between residue 1635-1636 located in one of the flexible loops of RTX block V using a previously described method.260 All of the constructed genes were confirmed by DNA sequencing. The protein sequences of these constructs are shown in Appendix. Protein expression and purification were performed using the same procedure as described in chapter 2 (2.5.1). SDS-PAGE was performed to confirm the molecular weight of the engineered proteins. SDS-PAGE confirmed the expression and purity of the engineered proteins (Figure 4.13). 117 Figure 4.13 Coomassie blue stained SDS-PAGE gel of different constructs of RTX block V. Lane 1, NuG2-wt RTX block V-NuG2 (34 kDa); Lane 2, NuG2-RTX Δhelix1529,1664-NuG2 (32 kDa); Lane 3, standard protein marker; Lane 4, NuG2-RTX block V W1645G-Y1646G-NuG2 (34 kDa); Lane 5, NuG2-RTX block V-L5-NuG2 (34 kDa); Lane 6, NuG2-RTX block V A1615C (28 kDa). 4.6.2 Preparation of DNA-protein Chimera. DNA handles and DNA-protein chimera were prepared using the same procedure as described in chapter 2 (2.5.2). 4.6.3 Single-Molecule OT Experiment. The single-molecule OT experiments (Figure 4.1B) were carried out in the same way as described in chapter 2 (2.5.3). 4.6.4 Measuring Force-Extension Relationship of Protein Unfolding The force-extension relationship of protein unfolding was measured in the same way as described in chapter 2 (2.5.4). 118 4.6.5 Calculating the Unfolding and Folding Kinetics. The unfolding and folding rate constants were calculated in the same way as described in chapter 3 (3.6.5). 119 Chapter 5: Single-Molecule Force Spectroscopy Reveals that the Mechanical Stability of Translocated RTX Domain Is Further Enhanced as the Secretion Proceeds 5.1 Synopsis CyaA is a virulent protein toxin, which needs to be secreted from the bacterial cytosol to the extracellular environment to function. Five consecutive RTX domains located at the C-terminus of CyaA can fold into unique β-roll structures by binding Ca2+ ions. We have used SMFS to provide new invaluable insights into the translocation mechanism of RTX block V, the first RTX domain to be secreted and folded. To obtain a bigger picture of how RTX domains in CyaA behave in the secretion process, here we employed OT to investigate the mechanical properties of another RTX domain, RTX block IV at the single-molecule level. Our results revealed that the apo-RTX block IV behaves as a random coil. Upon binding of Ca2+ ions, RTX block IV can fold into a mechanically stable structure on the folded RTX block V. The unfolding and folding of holo-RTX block IV mostly occurs via a four-state pathway. Our results showed that the mechanical stability is progressively enhanced from the N- to C-terminus throughout RTX block IV. Moreover, our studies suggested a strong interplay between RTX block IV and V, which stabilizes the folded RTX block V. Our results also highlighted the critical importance of the mechanical design for the RTX domains, which can effectively stabilize the toxin in vivo. 5.2 Introduction CyaA is produced by Gram-negative bacteria Bordetella pertussis.235-238 CyaA is a key virulence factor of whooping cough, which penetrates through the cytoplasmic membrane of host 120 cells and converts cytosolic ATP to cAMP at an uncontrolled level.111-112, 247 Understanding the molecular mechanism via which the toxin interacts with the host cells and ablates the physiological functions represents a significant challenge. CyaA bears five consecutive RTX domains at its C-terminus.239-240, 245 Each RTX domain consists of a series of glycine- and aspartate-rich nonapeptide repeats with a consensus sequence GGXGXDXXX (where X represents any amino acid), which are arranged in a tandem fashion and can fold into a right-handed parallel β-roll structure by binding Ca2+ ions.241, 249, 261 This unique structure has been previously reported to facilitate the secretion of CyaA and be responsible for binding CD11b/CD18 integrin.111, 246, 262 Once synthesized in the cell cytosol, where the concentration of Ca2+ is low (lower than 100 nM), RTX domains remain unstructured. After being translocated via the T1SS to the Ca2+-rich extracellular milieu (higher than 2 mM), the polypeptide can bind Ca2+ ions and fold into a mechanically stable structure.111, 245-246, 261-262 Using SMFS via OT, our previous work has revealed that the mechanical design of RTX block V governs its efficient translocation via the T1SS machinery.263 Three factors mainly contribute to the efficient translocation of RTX block V. (1) The apo-RTX block V behaves as a random coil, and can be threaded into the narrow channel of the T1SS without additional consumption of energy; (2) the translocated polypeptide can quickly fold into a stable structure, which behaves as a roadblock and prevents backsliding; (3) the folding of the translocated polypeptide can generate a further stretching force to actively facilitate the translocation by pulling the remaining polypeptide in the translocation conduit. However, after the translocation of RTX block V, it remains unclear whether the other RTX domains follow a similar mechanism to be translocated. Moreover, it is also worth investigating whether an interaction 121 exists between adjacent RTX domains. Here, we used OT to investigate the mechanical unfolding and folding of RTX block IV (residues 1377-1528). The 3D structure of RTX block IV-V (PDB: 6SUS) is shown in Figure 5.1A. RTX block IV consists of one N-terminal unfolded polypeptide and a β-roll structure composed of 12 β strands and 7 bound Ca2+ ions (Figure 5.1A, colored in red). The Ca2+ ions are maintained within the two neighboring peptide turns by charge-dipole interactions. The β strands are arranged in a tandem fashion and packed tightly onto the N-terminus of RTX block V (Figure 5.1A, colored in cyan). Overall, RTX block IV and V together yields a seemingly large contiguous β-roll structure and a C-terminal α helix.111, 264 122 Figure 5.1 Probing the unfolding and folding of RTX block IV using OT. (A) The 3D structure of RTX block IV-V (PDB: 6SUS). RTX block IV is composed of an unstructured polypeptide at the N-terminus and a β-roll structure with bound Ca2+ ions (residues 1377-1528, colored in red). RTX block V includes a Ca2+-binding β-roll structure and a C-terminal α helix (residues 1529-1681, colored in cyan). The Ca2+ ions are shown in grey spheres. (B) Schematics of the experimental OT setup. A protein chimera NuG2-RTX block IV-V-NuG2 is coupled with two DNA handles via thiol-maleimide chemistry. The flanked NuG2 domains serve as fingerprints for identification of the successful capture of a single-molecule. The DNA-protein complex is then held by two polystyrene beads, with streptavidin and anti-digoxigenin modified on their surface, respectively. One bead is sucked onto a glass micropipette and the other is controlled by the optical trap. By moving the position of laser trap, the target protein can be mechanically stretched or relaxed. During this process, the unfolding and folding of target protein can be directly observed. OT has been widely used to characterize the protein mechanical properties at the single-molecule level. Here, we employed OT to investigate the mechanical unfolding and folding of RTX block IV. Our results revealed that the folding of RTX block V is an essential step for the folding of RTX block IV. Our results also revealed that RTX block V is dramatically stabilized in 123 the presence of a the folded RTX block IV, suggesting a strong interaction between these two adjacent RTX domains. These mechanistic insights obtained by single-molecule study might offer new strategies for therapeutics of the bacterial-causing diseases. 5.3 Results 5.3.1 RTX Block IV Does Not Fold Independently. RTX block V is located at the very end of C-terminus of CyaA and is the first RTX domain to be translocated via the T1SS. The other RTX domains, in the order from block IV-I, will be translocated and folded in a successive order.111 To understand the molecular mechanism behind this secretion pattern, we investigated the conformational and mechanical properties of RTX block IV in both apo- and holo-state. To better determine the mechanical unfolding and folding events of RTX block IV, we constructed the polyprotein, NuG2-RTX block IV-NuG2 for OT experiments, where NuG2 is a well-characterized protein domain to serve as a fingerprint for identifying single-molecule capturing events. The mechanical unfolding of NuG2 generates a ΔLc of ~18 nm, and unfolding forces between 30-50 pN at a pulling speed of 50 nm/s.144, 206 In a given force-distance curve, the observation of the typical unfolding events of NuG2 domains indicates a successful single-molecule stretching event of the polyprotein, and ensures that the force-distance curve also contains mechanical signatures of RTX block IV. In the absence of Ca2+, stretching and relaxing the NuG2-apo-RTX block IV-NuG2 only generates a pair of unfolding and refolding events of NuG2 with a ΔLc of ~18 nm. No mechanical rupture event of RTX block IV is observed (Figure 5.2, left curve). This result is consistent with our previous investigations on RTX block V by SMFS,263 suggesting that the apo-RTX block IV behaves as a random coil and is mechanically compliant. 124 Figure 5.2 Representative force-distance curves showing the unfolding/folding of NuG2-apo-RTX block IV-NuG2 (left) and NuG2-apo-RTX block IV-V-NuG2 (right) at a pulling speed of 50 nm/s. Only the unfolding and refolding of NuG2 domains can be observed, while the mechanical signatures of RTX block IV and RTX block IV-V are missing, suggesting that RTX block IV and V behave as random coils at the absence of Ca2+ ions and do not display any mechanical resistance. To investigate the mechanical unfolding and folding of holo-RTX block IV, we carried out OT experiments in the presence of 10 mM Ca2+. As shown in Figure 5.3, the presence of two unfolding/folding events of NuG2 indicates the successful capture of a single molecule. However, the mechanical signature of RTX block IV is still missing. Clearly, even though supplemented with Ca2+ ions, RTX block IV still remains unstructured and does not show any mechanical resistance. This result shows that RTX block IV cannot fold independently, strongly suggesting that in addition to Ca2+, other factors govern the folding of RTX block IV to a mature structure. 125 Figure 5.3 Representative curves showing the unfolding/folding of NuG-RTX block IV-NuG2 at the pulling speed of 50 nm/s. The polyprotein was stretched and relaxed in the Tris buffer supplemented with 10 mM Ca2+. Two unfolding (indicated by grey circle) and refolding events (indicated by black circle) of fingerprints NuG2 domains are clearly observed, but the signatures of the mechanical unfolding and folding of RTX block IV are missing, suggesting that RTX block IV alone does not bind Ca2+ ions to fold and is mechanically labile. 5.3.2 Folded RTX Block V Is a Crucial Prerequisite for Folding of RTX Block IV. Since the translocation and folding of RTX block V precedes those of other RTX domains, it is plausible that the pre-folded β-roll of RTX block V in the extracellular milieu might facilitate the folding of RTX block IV. To mimic the successive folding of RTX block IV and V, we designed NuG2-RTX block IV-V-NuG2, and used OT to characterize its mechanical properties in both apo- and holo-form. Similarly, stretching apo-RTX block IV-V does not generate any mechanical rupture events (Figure 5.2, right curve), suggesting that the apo-RTX block IV-V is also intrinsically disordered in the Ca2+-depleted cytoplasm. In contrast, stretching holo-RTX block IV-V yields the unfolding signatures of RTX block IV-V. Figure 5.4A inset shows a representative force-distance curve of a full-length NuG2-RTX block IV-V-NuG2 at a pulling speed of 50 nm/s. Evidently, in addition to the mechanical unfolding events of two NuG2 domains, 126 new unfolding events were observed at ~10 pN, which can be readily attributed to the unfolding of holo-RTX block IV-V. Since the unfolding forces of RTX block IV-V were significantly lower than those of NuG2, subsequent stretching-relaxation cycles were limited below 20 pN so that only RTX block IV-V was unfolded while NuG2 domains remained in the folded state. Figure 5.4A demonstrates two force-distance curves of holo-RTX block IV-V. During the stretching process, it is obvious that each force-distance curve shows two major rupture events (colored in red and green, respectively), suggesting that upon stretching, the unfolding of holo-RTX block IV-V occurs via a two-step fashion, in which RTX block IV and V may unfold separately. 127 Figure 5.4 Holo-RTX block IV-V is mechanically stable and can fold rapidly. (A) Representative force-distance curves showing the unfolding/folding behaviors of RTX block IV (colored in red) and V (colored in green). The pulling speed was 50 nm/s. Inset: a representative force-distance curve of the full-length NuG2-RTX block IV-V-NuG2. The unfolding and refolding events of NuG2 domains are colored in black and indicated by the triangles. The unfolding and refolding events of RTX block IV and RTX block V are indicated by circles and squares, respectively. (B) Representative force-distance curves of the unfolding/folding of RTX block IV via the four-state pathway. The pulling speed was 50 nm/s. Zoomed-in views showed that the unfolding and refolding of RTX block IV involve two intermediate states. The grey dashed lines in (A) and (B) were pseudo WLC fits of the force-distance curves, generated by our self-developed Igor Pro program. (C) Force-extension relationships of the unfolding of RTX block IV-V. The dashed lines are the WLC fits to the experimental data for RTX block IV from N→I1 (yellow), I1→I2 (blue), I2→U (black), N→U (red), respectively, and for RTX block V from N→U (green). The WLC fits yielded the persistence length of 0.8 nm and ΔLc1 of 6.5 ± 0.9 nm, ΔLc2 of 11.3 ± 0.7 nm, ΔLc3 of 23.5 ± 0.5 nm, ΔLc IV of 39.9 ± 0.8 nm and ΔLc V of 46.2 ± 0.9 nm, respectively. (D) Histograms of unfolding and refolding forces of RTX block IV at a pulling speed of 50 nm/s. 128 Fitting the length changes at different forces of the two unfolding events to the WLC model measures a contour length increment ΔLc of 39.9 ± 0.8 nm (Figure 5.4C, colored in red) and 46.2 ± 0.9 nm (Figure 5.4C, colored in green), respectively. As revealed by our previous SMFS studies on holo-RTX block V, the ΔLc of 46.2 nm is in good agreement with the ΔLc for the two-state unfolding of holo-RTX block V,263 thus the second unfolding event (Figure 5.4A, colored in green) can be assigned to the mechanical unfolding of RTX block V. Zoomed-in view of the second unfolding event also shows the intermediate state of the unfolding of holo-RTX block V (Figure 5.5), which further corroborates that RTX block V unfolds independently after the unfolding of RTX block IV. Taken together, the first unfolding event can be readily attributed to the unfolding of holo-RTX block IV (Figure 5.4A, colored in red). Figure 5.5 Zoomed-in view of the second unfolding event of RTX block IV-V. An unfolding intermediate state is observed. The contour length increments are measured as ΔLc1 of ~17 nm and ΔLc2 of ~28 nm, suggesting that RTX block V unfolds after RTX block IV has unfolded. RTX block IV spans residues 1377-1528 and possesses ten RTX motifs. In the crystallographic structure of RTX block IV, only a C-terminal β-roll consisting of seven RTX motifs (residues 1410-1528) is displayed, while the rest of the RTX motifs at the N-terminus (residues 1377-1409) are missing.264 If the three predicted RTX motifs also fold into a β-roll 129 structure, complete unfolding of holo-RTX block IV would give rise to a ΔLc of ~50 nm (152 aa × 0.36 nm/aa – 4.5 nm = 50.2 nm, where 4.5 nm is the assumed distance between the N- and C-termini of RTX block IV), which is about 10 nm longer than the experimentally measured ΔLc. This result suggested that the three RTX motifs are mechanically compliant and do not generate any mechanical signatures during a stretching process. Unfolding of the folded β-roll structure in RTX block IV (residues 1410-1528) would result in a ΔLc of 40.4 nm (119 aa × 0.36 nm/aa – 2.4 nm = 40.4 nm, where 2.4 nm is the distance between I1410 and V1528), in good agreement with the experimentally measured ΔLc. To verify this assignment, we constructed two variants of RTX block IV-V (S1396C and T1411C) by site-directed mutagenesis (termed as RTX block IV1396,1528-V and RTX block IV1411,1528-V). Residue Ser1396 is located at the N-terminal region of the predicted RTX motifs, and residue Thr1411 is located at the N-terminal end of the β-roll of the crystal structure. If the three N-terminal RTX motifs are indeed unstructured, we expect that the ΔLc will not be affected in these two variants. As shown in Figure 5.6 and 5.7, stretching RTX block IV-V from residue Ser1396 (or Thr1411) and the C-terminus generates the force-distance curves similar to those of wt RTX block IV-V. The WLC fitting to the length changes at different forces also measures an identical ΔLc to that of wt RTX block IV-V. This result suggested that the observed unfolding event in the single-molecule stretching experiments indeed originates from the unfolding of the folded β-roll, and that the unfolding of three N-terminal RTX motifs occurs at low forces and does not yield a clear unfolding signature. 130 Figure 5.6 Mechanical unfolding/folding of RTX block IV1396,1528-V. (A) Schematics of stretching RTX block IV1396,1528-V from the C-terminus and Ser1396 (indicated by the green asterisk). (B) Representative force-distance curves showing the unfolding/folding of RTX block IV1396,1528 via a four-state pathway at a pulling speed of 50 nm/s. (C) The WLC fitting of force-extension relationships of the unfolding of RTX block IV1396,1528 from N→I1 (yellow), I1→I2 (blue), I2→U (black) and N→U (red), respectively. The WLC fits yielded a persistence length of 0.8 nm and ΔLc1 of 6.0 ± 0.3 nm, ΔLc2 of 12.1 ± 0.2 nm, ΔLc3 of 21.6 ± 0.5 nm and ΔLc of 39.3 ± 0.4 nm, respectively. 131 Figure 5.7 Mechanical unfolding/folding of RTX block IV1411,1528-V. (A) Schematics of stretching RTX block IV1411,1528-V from the C-terminus and Thr1411 (indicated by the green asterisk). (B) Representative force-distance curves showing the unfolding/folding RTX block IV1411,1528-V at a pulling speed of 50 nm/s. The unfolding/folding of RTX block IV1411,1528 are colored in red, and the unfolding/folding of RTX block V is colored in green. The unfolding always initiates from the unfolding of RTX block IV1411,1528, and folding initiates from the folding of RTX block V. (C) The WLC fitting of force-extension relationships of the unfolding of RTX block IV1411,1528 (red) and RTX block V (green), respectively. The WLC fits yielded a persistence length of 0.8 nm and ΔLc1 (RTX block IV1411,1528) of 38.3 ± 0.5 nm, and ΔLc2 (RTX block V) of 44 ± 1 nm, respectively, in good agreement with the theoretical calculations. (RTX block IV1411, 1528: 118 aa × 0.36 nm/aa- 2.3 nm = 40.2 nm, where 2.3 nm is the distance between T1411 and V1528. RTX block V: 136 aa × 0.36 nm/aa − 3.2 nm = 45.8 nm, where 3.2 nm is the distance between G1529 and D1664). Taken together, RTX block IV can fold into a β-roll structure on the folded RTX block V in the presence of 10 mM Ca2+. Stretching the folded RTX block IV-V can generate a sequential 132 unfolding, in which the unfolding of RTX block IV always precedes the unfolding of RTX block V. Considering the fact that RTX block IV does not fold independently, it is likely that the complete folded structure of RTX block V is important to the folding of RTX block IV. 5.3.3 The Mechanical Unfolding of RTX Block IV Involves the Formation of Multiple Intermediate States. Since RTX block IV tends to unfold before RTX block V, we stretched RTX block IV-V until RTX block IV unfolded and then relaxed the molecule immediately. In this way, we only allowed RTX block IV to unfold, facilitating the observation of unfolding/folding of RTX block IV. Figure 5.4B shows a pair of such force-distance curves that only show the unfolding of RTX block IV. A zoomed-in view of the unfolding process reveals the formation of two metastable intermediate states, indicating that the unfolding occurs via a four-state pathway. The WLC fits to the extensions at different forces yielded the ΔLc1 of 6.5 nm, ΔLc2 of 11.3 nm and ΔLc3 of 23.5 nm, respectively (N→I1, I1→I2 and I2→U, Figure 5.4C). The sum of ΔLc1 to ΔLc3 equals to 41.3 nm, in excellent agreement with the ΔLc obtained from the complete unfolding of holo-RTX block IV. It is of note that RTX block IV can also unfold via different pathways. In some scenarios, either one or both of the two short-lived intermediate state(s) might be skipped during an unfolding process, leading to a three-state or two-state unfolding pathway. Nevertheless, the majority of the unfolding still occurs via the four-state pathway, accounting for ~96% of all the unfolding pathways (Table 5.1). Table 5.1 Percentage of each pathway via which the unfolding/folding of RTX block IV occurs N-I1-I2-U N-I1-U N-I2-U N-U Unfolding 96.4% 0.6% 2.9% 0.2% Folding 70.6% 4.0% 19.4% 6.0% 133 The average unfolding force of RTX block IV at the pulling speed of 50 nm/s is ~10 pN (Figure 5.4D and Table 5.2), suggesting that the holo-RTX block IV is mechanically stable. Using the method proposed by Oesterhelt et al.,140 we measured the force-dependent unfolding rate constants for each unfolding step in the four-state pathway (Figure 5.8). Fitting the force-dependent unfolding rate constants to the Bell-Evans model yielded the intrinsic unfolding rate constant at zero force (α0) and the unfolding distance (Δxu) (Table 5.2). It is of note that the unfolding rate constant for I1→I2 ((2.4 ± 0.7) × 10-1 s-1) or I2→U (2.7 ± 0.3 s-1) was 2-3 order of magnitude higher than that for N→I1 ((1.6 ± 0.3) × 10-3 s-1), implying that the intermediate state was rather mechanically unstable. Table 5.2 Average unfolding and folding forces and kinetic parameters for RTX block IV Fu (pN) Ff (pN) α0 (s-1) β0 (s-1) Δxu (nm) Δxf (nm) RTX block IV N-I1 10 ± 1 (n=505) 5.0 ± 0.6 (n=389) (1.6 ± 0.3) × 10-3 (1.6 ± 0.1) × 103 3.4 ± 0.2 2.5 ± 0.2 I1-I2 10 ± 1 (n=502) 4.6 ± 0.6 (n=368) (2.4 ± 0.7) × 10-1 (1.8 ± 0.3) × 102 1.8 ± 0.2 1.8 ± 0.6 I2-U 10 ± 1 (n=517) 4.1 ± 0.6 (n=469) 2.7 ± 0.3 (7.1 ± 0.6) × 103 0.3 ± 0.1 7.8 ± 0.6 134 Figure 5.8 Force-dependency of the unfolding (red) and folding (blue) rate constants of RTX block IV for the pathways of (A) N↔I1, (B) I1↔I2 and (C) I2↔U. The rate constants were calculated using the Oesterhelt method and the fitting of dwell-time distribution. Dashed lines correspond to the fits of Bell-Evans model to the experimental data. Kinetic parameters are summarized in Table 5.2. 5.3.4 Unfolded RTX Block IV Can Fold Back to the Native State Following Parallel Pathways. After the mechanical unfolding of RTX block IV-V, the folded β-roll structure is converted into an unfolded polypeptide, accompanied by the dissociation of Ca2+ ions. To investigate the folding behaviors, we relaxed the unfolded RTX block IV-V to allow for protein refolding. Upon relaxation, the unfolded RTX block IV-V was observed to fold into the native state via an intermediate state (Figure 5.4A and D and Table 5.2). Subsequent stretching resulted in an identical unfolding pattern of the unfolding of RTX block IV-V, suggesting that RTX block IV-V can refold efficiently to its native structure against a stretching force. The observed two ΔLc for the two-step folding of RTX block IV-V are the same as those for the two-step unfolding, suggesting that the folding of RTX block IV-V occurs in the order of RTX block V-IV (Figure 5.4A). A zoomed-in view in Figure 5.4B illustrates that the folding of RTX block IV also involves the formation of intermediate states. A large portion of RTX block IV folds via a four-state pathway (70.6%), while a small portion folds in a two-state (6%) or three-state (23.4%) fashion. It 135 is also of note that due to the insufficient time resolution, the intermediate states during the folding process might not be well resolved, and the two- or three-state pathway could be just a special case of the four-state pathway. The contour length changes observed in the four-state folding process are equivalent to those in the four-state unfolding process, suggesting that the two intermediate states in both unfolding and folding of RTX block IV are structurally similar (if not the same). Fitting the force-dependency of the folding rate constants to the Bell-Evans model also measured the intrinsic folding rate constant at zero force (β0) and the folding distance (Δxf) (Table 5.2). It is important to note that the intrinsic folding rate constant of RTX block IV is high (with β0 of 7.1 × 103 s-1) and can be compared with the folding rate constant of RTX block V (with β0 of 8.2 × 103 s-1).263 5.3.5 The Unfolding/Folding Mechanism of RTX Block IV. The observation of the intermediate states during the unfolding of holo-RTX block IV suggested that two partially folded structures were able to exist under a high stretching force albeit short-lived. To unveil the structures of the intermediate states, we constructed another variant of RTX block IV-V (A1429C) (termed as RTX block IV1429,1528-V). Amino acid residue Ala1429 is located at the second β stand of the β-roll structure. Stretching RTX block IV-V from Ala1429 and the C-terminus leads to the unfolding of part of the β-roll structure in RTX block IV-V, while the N-terminal part of the β-roll beyond Ala1429 is not subject to stretching (Figure 5.9A). Hence, stretching this variant will lead to a total ΔLc shortening by ~7 nm (the expected ΔLc for the unfolding of the N-terminal part). If the unfolding of RTX block IV initiates from the N-terminus, stretching RTX block IV1429,1528 will result in a disappearance of ΔLc1 (~6.5 nm) for the four-state unfolding of wt RTX block IV. Therefore, the unfolding of RTX block IV1429,1528 will occur via a three-state pathway. 136 As shown in Figure 5.9B and C, stretching RTX block IV1429,1528 resulted in force-distance curves showing a clear three-state unfolding of RTX block IV1429,1528. The WLC fitting to the force-extension data measured the ΔLc1 and ΔLc2 for the three-state unfolding of RTX block IV1429,1528, which were identical to the ΔLc2 and ΔLc3 for the four-state unfolding of wt RTX block IV. This result suggested that the first unfolding event observed in wt RTX block IV can be assigned to the unfolding of the N-terminal part of the β-roll structure, and the unfolding of RTX block IV initiates from the N-terminus. Similar to wt RTX block IV, the folding pathway of RTX block IV1429,1528 also appears to be the reverse of the unfolding pathway, i.e., the folding events occurred in the order of the event with ΔLc2 followed by the event with ΔLc1, suggesting that the folding of RTX block IV starts from the C-terminus. 137 Figure 5.9 Unfolding/folding of RTX block IV1429,1528 occurs via a three-state pathway. (A) Schematics of stretching RTX block IV1429,1528-V from its C-terminus and Ala1429 (indicated by the green asterisk). (B) Representative force-distance curves showing the unfolding/folding of RTX block IV1429,1528-V at a pulling speed of 20 nm/s. Zoomed-in views showed that the unfolding (colored in red) and refolding (colored in blue) of RTX block IV1429,1528 involve an intermediate state. The unfolding/folding of RTX block V are colored in green. (C) The WLC fitting of force-extension relationships of the unfolding of RTX block IV1429,1528 from N→I (red), and I→U (blue), respectively. The WLC fits yielded a persistence length of 0.8 nm and ΔLc1 of 11.5 ± 0.2 nm, and ΔLc2 of 22.7 ± 0.3 nm, respectively. Putting together all the structural and mechanical information obtained from the above OT studies, a detailed picture of the holo-RTX block IV unfolding/folding mechanism can be obtained (Figure 5.10). Upon stretching, RTX block IV is unfolded from the N-terminus, mostly via a four-state unfolding pathway (~96%). In the first step, one β strand at the very end of the N-terminus 138 as well as random coiled polypeptide (from Gly1377 to Val1427, 18 aa × 0.36 nm/aa – 0.6 nm = 5.9 nm) is unfolded and extended (Figure 5.10, colored in red). Four neighboring β strands (from Leu1428 to Trp1463, 36 aa × 0.36 nm/aa – 1.0 nm = 12.0 nm) are subsequently unfolded from the β-roll structure (Figure 5.10, colored in yellow), followed by the unfolding of the remaining 7 β strands (from Phe1464 to Val1528, 65 aa × 0.36 nm/aa – 1.0 nm = 22.4 nm) at the C-terminus of RTX block IV (Figure 5.10, colored in green). Besides the dominant four-state unfolding pathway, a small fraction of the proteins might skip either one or both intermediate state(s) to the unfolded state, resulting in a three- or two-state unfolding pathway (Figure 5.10, indicated by dashed arrows). 139 Figure 5.10 Schematics showing the unfolding and folding pathways of RTX block IV. In the four-state unfolding pathway N→I1→I2→U, the N-terminal unfolded polypeptide as well as one β strand (colored in red) will be extended and unfolded first. Then four β strands (colored in yellow) will be stretched and unfolded next, followed by the unfolding of the rest of RTX block IV structure (colored in green). RTX block V is colored in cyan. The four-state folding of RTX block IV will follow a reverse order of the unfolding, which is U→I2→I1→N. Dashed curves represents that RTX block IV can also unfold/fold via a three-state (N↔I1↔U, N↔I2↔U) or a two-state pathway (N↔U). The percentage of each unfolding and folding pathway is shown in Table 5.1. In the folding process, RTX block IV folds from the C-terminus. The β strands are packed one by one onto the N-terminus of RTX block V to form the native β-roll structure. A large portion of RTX block IV (~70%) follows a parallel four-state folding pathway to fold back to its native structure, while a small portion folds using the three-state or two-state path. 140 5.3.6 RTX Block IV-V Displays a Different Mechanical Stability Between Its N- and C-terminus. The unfolding of RTX block IV occurs following a strict order, N→I1→I2→U. Not even a single RTX block IV was observed to unfold in a different fashion, e.g., N→I2→I1→U, despite that the average unfolding forces for the three steps were rather close (N→I1: 9.8 pN, I1→I2: 9.7 pN, I2→U: 9.8 pN). Moreover, the unfolding of RTX block IV always precedes that of RTX block V, while the average unfolding force of RTX block IV (~10 pN) was not significantly lower than that of RTX block V (~14 pN). These observations are especially important considering that the unfolding of tandem modular proteins consisting of multiple domains is strictly dependent upon their mechanical stability. For unfolding events with similar unfolding forces, it is expected that there will be no strict unfolding hierarchy. The observed strict unfolding order/hierarchy is an inherent feature of RTX block IV-V, and different parts of RTX block IV-V have a hierarchical stability, so does RTX block IV and RTX block V. This implies that the mechanical stability of the intermediate states and RTX block V might be enhanced by the N-terminal folded β-roll structure. To unfold the intermediate states or RTX block V directly from RTX block IV-V, a higher stretching force is expected to be required. To test this hypothesis, we used OT to stretch RTX block IV1429,1528-V and measure its unfolding forces (Figure 5.9A and 5.11). As shown in Figure 5.11A, the unfolding of RTX block IV1429,1528-V showed a complex picture. In most cases (~96%), unfolding of RTX block IV1429,1528-V initiates from RTX block IV1429,1528. Figure 5.9C and 5.11A (left curve) show the unfolding/folding curves for such molecules. Fitting the length changes at different forces of the two unfolding events to the WLC model results in a contour length increment ΔLc1 of 32.9 ± 0.2 nm (Figure 5.11B, colored in red) and ΔLc2 of 43.2 ± 0.6 nm (Figure 5.11B, colored in green), 141 respectively. Unfolding of RTX block IV1429,1528 would theoretically result in a ΔLc of 34.1 nm (100 aa × 0.36 nm/aa - 1.9 nm = 34.1 nm, where 1.9 nm is the distance between A1429 and V1528), in good agreement with the experimentally measured ΔLc1. This result suggested that RTX block IV1429,1528 unfolds before RTX block V. In contrast, a very small fraction (~4%) was observed to unfold from RTX block V (Figure 5.11A, right curve). Fitting the force-extension relationship to the WLC model yields a ΔLc1 of ~44 nm and ΔLc2 of ~33 nm, suggesting that the unfolding of RTX block V takes place as a first step. Figure 5.11C shows the unfolding force histograms of RTX block IV1429,1528-V. The histograms are colored in red or green depending on which part of RTX block IV1429,1528-V unfolds first. The average unfolding force of RTX block IV1429,1528-V at a pulling speed of 50 nm/s is 20.1 pN, higher than that of wt RTX block IV-V. This result suggested that the unfolding of part of the C-terminal β-roll structure requires a higher stretching force. 142 Figure 5.11 Mechanical unfolding/folding of RTX block IV1429,1528-V. (A) Representative force-distance curves showing the unfolding/folding of RTX block IV1429,1528-V at a pulling speed of 50 nm/s. The unfolding/folding of RTX block IV1429,1528 is colored in red, and the unfolding/folding of RTX block V is colored in green. The unfolding initiates from either RTX block IV or block V, but folding always starts from RTX block V. (B) The WLC fitting of the force-extension relationships of the unfolding of RTX block IV1429,1528 (red) and RTX block V (green), respectively. The WLC fits yielded a persistence length of 0.8 nm and ΔLc1 (RTX block IV1429,1528) of 32.9 ± 0.2 nm, and ΔLc2 (RTX block V) of 43.2 ± 0.6 nm, respectively, in good agreement with the theoretical calculations. (RTX block IV1429,1528: 100 aa × 0.36 nm/aa - 1.9 nm = 34.1 nm, where 1.9 nm is the distance between A1429 and V1528). (C) Unfolding force histogram of RTX block IV1429,1528-V. A large portion of RTX block IV1429,1528-V unfolds from RTX block IV1429,1528 (~96%, colored in red), while a small portion unfolds from RTX block V (~4%, colored in green). To further corroborate this hypothesis, we engineered three more variants of RTX block IV-V (S1447C, F1465C and T1483C, termed as RTX block IV1447,1528-V, RTX block IV1465,1528-V, RTX block IV1483,1528-V). Amino acids Ser1447, Phe1465 and Thr1483 are located at the fourth, 143 sixth and eighth β strand of the β-roll, respectively (Figure 5.12-14). Stretching RTX block IV-V from Ser1447 (or Phe1465, Thr1483) and the C-terminus also leads to the unfolding of only part of the β-roll structure. Similar to RTX block IV1429,1528-V, the unfolding of RTX block IV1447,1528-V displays two unfolding orders (Figure 5.12B). Upon stretching, a small percentage of RTX block IV1447,1528-V unfolds from RTX block IV1447,1528 (~25%), but a large fraction unfolds from RTX block V (~75%). The unfolding of RTX block IV1465,1528-V and RTX block IV1483,1528-V are different from that of RTX block IV1429,1528-V. As shown in Figure 5.13B and 5.14B, all the molecules of RTX block IV1465,1528-V and RTX block IV1483,1528-V are observed to unfold from RTX block V. As shown in Figure 5.12D, 5.13D and 5.14D, the average unfolding force of RTX block IV1447,1528-V, RTX block IV1465,1528-V and RTX block IV1483,1528-V at a pulling speed of 50 nm/s are 28.0 pN, 30.9 pN and 30.4 pN, respectively, significantly higher than that of wt RTX block IV-V. Taken together, these results indicated that the mechanical stability of C-terminal β-roll is enhanced by the N-terminal part. 144 Figure 5.12 Mechanical unfolding/folding of RTX block IV1447,1528-V. (A) Schematics of stretching RTX block IV1447,1528-V from its C-terminus and Ser1447 (indicated by the green asterisk). (B) Representative force-distance curves showing the unfolding and folding of RTX block IV1447,1528-V at a pulling speed of 50 nm/s. The unfolding/folding of RTX block IV1447,1528 is colored in red, and the unfolding/folding of RTX block V is colored in green. The unfolding starts from either RTX block IV or block V, but the folding always starts from RTX block V. (C) The WLC fitting of force-extension relationships of the unfolding of RTX block IV1447,1528 (red) and RTX block V (green), respectively. The WLC fits yielded a persistence length of 0.8 nm and ΔLc1 (RTX block IV1447,1528) of 28.7 ± 0.6 nm, and ΔLc2 (RTX block V) of 42.8 ± 0.5 nm, respectively, in good agreement with the theoretical calculations. (RTX block IV1447,1528: 82 aa × 0.36 nm/aa - 1.5 nm = 28.0 nm, where 1.5 nm is the distance between S1447 and V1528). (D) Unfolding force histogram of RTX block IV1447,1528-V. A small portion of RTX block IV1447,1528-V unfolds from RTX block IV1447,1528 (~25%, colored in red), while a large portion unfolds from RTX block V (~75%, colored in green). 145 Figure 5.13 Mechanical unfolding/folding of RTX block IV1465,1528-V. (A) Schematics of stretching RTX block IV1465,1528-V from its C-terminus and Phe1465 (indicated by the green asterisk). (B) Representative force-distance curves showing the unfolding/folding of RTX block IV1465,1528-V at a pulling speed of 50 nm/s. The unfolding/folding of RTX block IV1465,1528 is colored in red, and the unfolding/folding of RTX block V is colored in green. The unfolding and folding both initiate from RTX block V. (C) The WLC fitting of force-extension relationships of the unfolding of RTX block IV1465,1528 (red) and RTX block V (green), respectively. The WLC fits yielded a persistence length of 0.8 nm and ΔLc1 (RTX block IV1465,1528) of 22.5 ± 0.4 nm, and ΔLc2 (RTX block V) of 42.5 ± 0.6 nm, respectively, in good agreement with the theoretical calculations. (RTX block IV1465,1528: 64 aa × 0.36 nm/aa - 1.0 nm = 22.0 nm, where 1.0 nm is the distance between F1465 and V1528). (D) Unfolding force histogram of RTX block IV1465,1528-V. All the molecules are observed to unfold from RTX block V (colored in green). 146 Figure 5.14 Mechanical unfolding/folding of RTX block IV1483,1528-V. (A) Schematics of stretching RTX block IV1483,1528-V from its C-terminus and Thr1483 (indicated by the green asterisk). (A) Representative force-distance curves showing the unfolding/folding of RTX block IV1483,1528-V at a pulling speed of 50 nm/s. The unfolding/folding of RTX block IV1483,1528 is colored in red, and the unfolding/folding of RTX block V is colored in green. The unfolding and folding both initiate from RTX block V. (B) The WLC fitting of the unfolding of force-extension relationships of the unfolding of RTX block IV1483,1528 (red) and RTX block V (green), respectively. The WLC fits yielded a persistence length of 0.8 nm and ΔLc1 (RTX block IV1483,1528) of 16.1 ± 0.3 nm, and ΔLc2 (RTX block V) of 43.1 ± 0.3 nm, respectively, in good agreement with the theoretical calculations. (RTX block IV1483,1528: 46 aa × 0.36 nm/aa - 0.7 nm = 15.9 nm, where 0.7 nm is the distance between T1483 and V1528). (C) Unfolding force histogram of RTX block IV1483,1528-V. All the molecules are observed to unfold from RTX block V (colored in green). By stretching RTX block IV-V via different stretching sites (Figure 5.15A), we found that the average unfolding force of RTX block IV-V is dependent on the stretching site located at the β-roll structure. As shown in Figure 5.15B, the average unfolding force varies as the stretching site 147 moves from the N-terminus to the C-terminus. There are three stages for the change of the average unfolding force: Stage (1): when the stretching site is located at the unfolded part of RTX block IV (Figure 5.11A, circled number 1 and 2), the average unfolding force remains almost unchanged compared with that of wt RTX block IV-V (9.8 pN); Stage (2): when the stretching site is located at the N-terminus of the β-roll structure (Figure 5.11A, circled number 3-5), the average unfolding force increases linearly as a function of the number of amino acid residues that is not subject to stretching; Stage (3): when the stretching site is located at the C-terminus of the β-roll structure (Figure 5.11A, circled number 6-7), the average unfolding force stops rising and remains almost unchanged. 148 Figure 5.15 RTX block IV-V displays a different mechanical stability between its N- and C-terminus. (A) Schematics showing the stretching of RTX block IV-V from its C-terminus and 7 different stretching sites (G1377, S1396C, T1411C, A1429C, S1447C, F1465C and T1483C) at the N-terminus (indicated by the green asterisk and circled numbers 1-7). The black dashed line divides RTX block IV into two parts: the N-terminal unfolded polypeptide and the C-terminal β-roll structure. (B) Relationships between the average unfolding force of different variants of RTX block IV-V and the number of amino acid residues that is not subject to stretching. All protein mutants are stretched at 3 different pulling speed of 5, 20 and 50 nm/s. (C) Percentage of different unfolding pathways (Two-state, RTX block IV-RTX block V and RTX block V-RTX block IV) of the 7 RTX block IV-V constructs obtained at a pulling speed of 5, 20 and 50 nm/s. 149 These results showed that RTX block IV-V displays unique mechanical properties. Firstly, stage (1) shows that stretching RTX block IV-V from its N-terminal unstructured polypeptide and C-terminal end does not lead to a higher unfolding force, suggesting that the unstructured polypeptide does not stabilize the folded β-roll structure; secondly, the increasing trend of the average unfolding force revealed in stage (2) suggests that the mechanical stability of the β-roll is progressively increased from the N- to the C-terminus; thirdly, stage (3) reveals an upper limit (~30 pN at a pulling speed of 50 nm/s) for the increasing trend of the average unfolding force, implying that the mechanical stability remains almost unchanged at the C-terminus of the β-roll. Overall, our studies revealed that RTX block IV-V displays distinct mechanical stabilities at its N- and C-terminus. Stretching RTX block IV-V from its N- and C-terminal end requires a low force of ~10 pN and induces the unfolding of RTX block IV in a successive manner. In contrast, it requires a higher force to directly stretch and unfold a part of the β-roll structure (up to ~30 pN) while its N-terminal part is kept intact. These results suggested an uneven distribution of the mechanical stability throughout RTX block IV-V. 5.3.7 RTX Block IV and V Can Interact and Stabilize Each Other. Our studies on these different variants of RTX block IV revealed a reversed unfolding hierarchy from RTX block V to IV. As shown in Figure 5.11-14, the unfolding of RTX block V could occur in the first place, followed by rapid unfolding of the stretched RTX block IV. Figure 5.15C shows the percentage of each unfolding pathway at different pulling speeds. If the stretching site is near the C-terminus of RTX block IV, the unfolding of RTX block V will take place as a first step. The unfolding hierarchies of the stretched RTX block IV and V are dependent upon their mechanical stability. For unfolding of RTX block IV1429,1528-V and RTX block IV1447,1528-V, the unfolding initiated from either RTX block IV or V, suggesting that the mechanical stability of the 150 stretched β-roll structure in RTX block IV is comparable to that of RTX block V; for unfolding of RTX block IV1465,1528-V and RTX block IV1483,1528-V, the unfolding always initiated from RTX block V, suggesting that the stretched β-roll structure in RTX block IV is much more stable than that in RTX block V. This reversed unfolding hierarchy allowed us to investigate the unfolding of RTX block V when RTX block IV is still folded. As shown in Figure 5.11-14, when RTX block V unfolds before RTX block IV, the average unfolding force was ~30 pN, significantly higher than that of the wt RTX block V (~14 pN), indicating that the neighboring N-terminal β-roll structure can stabilize RTX block V. With the reversed unfolding pattern of different RTX block IV-V variants, we can now measure the unfolding kinetics of RTX block V in the presence of a folded RTX block IV. Fitting the force-dependency of the unfolding rate constants to Bell-Evans model yielded the intrinsic unfolding rate constant at zero force (α0) of ~1.0 × 10-4 s-1 (Figure 5.16), much smaller than that of wt RTX block V (6.9 × 10-3 s-1), indicating much slower mechanical unfolding kinetics of RTX block V in the presence of a folded RTX block IV. This slower mechanical unfolding kinetics corresponds to a 4.2 kBT of stabilization of RTX block V by the folded RTX block IV. 151 Figure 5.16 Force-dependency of the unfolding rate constants of the unfolding of RTX block V when RTX block IV is folded. Fitting the force-dependency to the Bell-Evans model measured the unfolding distance Δxu of 1.3 nm and the intrinsic unfolding rate constants at zero force α0 of (1.5 ± 0.2) × 10-4 s-1, (1.1 ± 0.2) × 10-4 s-1 and (1.0 ± 0.1) × 10-4 s-1, obtained from the construct RTX block IV1447,1528-V, RTX block IV1465,1528-V, and RTX block IV1483,1528-V, respectively. The reversed unfolding hierarchy also allows us to measure the unfolding kinetics of the stretched part of RTX block IV when RTX block V is unfolded. Figure 5.17 (data points colored in blue) shows the force-dependent unfolding rate constants for unfolding RTX block IV1447,1528 in the absence of folded RTX block V. Fitting the force-dependency of the unfolding rate constants to the Bell-Evans model measured the intrinsic unfolding rate constant at zero force of ~2.9 × 10-1 s-1 and the unfolding distance of 0.7 nm. Since a small percentage of RTX block IV1447,1528-V unfolded from RTX block IV1447,1528 (~25%), stretching this construct also allows us to measure the unfolding kinetics of RTX block IV1447,1528 in the presence of folded RTX block V. The force-dependent unfolding rate constants are also shown in Figure 5.17 (data points colored in red). 152 Fitting the force-dependency of the unfolding rate constants to the Bell-Evans model yielded the intrinsic unfolding rate constant at zero force of ~1.8 × 10-2 s-1 (smaller than 2.9 × 10-1 s-1), indicating a slower unfolding kinetics of RTX block IV1447,1528 when RTX block V is folded. This result suggests that the β-roll in RTX block V can in turn stabilize RTX block IV. Figure 5.17 Force-dependency of the unfolding rate constants of the unfolding of RTX block IV1447,1528 when RTX block V is unfolded (blue) and folded (red). Fitting the force-dependency to the Bell-Evans model measured the unfolding distance Δxu of 0.7 nm and the intrinsic unfolding rate constants at zero force α0 of (2.9 ± 0.3) × 10-1 s-1 and (1.8 ± 1.1) × 10-2 s-1, respectively. It is of note that in the unfolding of RTX block IV1429,1528-V, RTX block IV1447,1528-V, RTX block IV1465,1528-V and RTX block IV1483,1528-V, if RTX block V unfolded first at ~30 pN, the stretched part of RTX block IV unfolded immediately. The short lifetime of the stretched part of RTX block IV suggested that when RTX block V is unfolded, the β-roll in RTX block IV becomes mechanically unstable and can be quickly unfolded under high tension. This result explains why stretching RTX block IV1465,1528-V and RTX block IV1483,1528-V leads to a similar average unfolding force at ~30 pN (Figure 5.15B). 153 Overall, our studies suggest that RTX block IV and V do interact with each other closely, leading to mutual stabilization of the two RTX domains. 5.4 Discussion 5.4.1 Folded RTX Block V Is Necessary for the Folding of RTX Block IV. The molecular design of RTX blocks at the C-terminus of CyaA is essential for its efficient translocation. Similar to previous studies on RTX block V, our single-molecule OT experiments on RTX block IV revealed the existence of multiple intermediate states in the folding of RTX block IV. This result suggested that the folding of RTX block IV also provides extra pulling forces to accelerate the translocation and make the whole process more efficient. Moreover, this study revealed that RTX block IV does not fold independently. Instead, the folding of RTX block IV requires the folded structure of RTX block V as its template. The secretion/translocation of CyaA is a critical step for CyaA to function and RTX block V is the first portion of CyaA to be secreted. Since the folding of RTX block IV depends on the folding of RTX block V, it is reasonable to speculate that any misfolding of RTX block V may lead to a failure of the secretion of CyaA. This result may offer new insights into the development of new therapeutic ways to ablate the toxic activities of CyaA by inhibiting the folding of RTX domains during secretion. 5.4.2 Folded RTX Block IV in Turn Stabilizes the Translocated RTX Block V. In the Ca2+-rich extracellular milieu, the translocated polypeptide chain of RTX block V can quickly fold into a mature structure, which serves as a roadblock preventing backsliding and providing an anchoring point for further translocation.263 The translocated RTX block V should be stable in the extracellular milieu. If RTX block V could be easily unfolded from the C-terminus, backsliding of the translocated polypeptide might occur due to the absence of the “roadblock”, 154 leading to a less efficient translocation. Therefore, the stability of folded β-roll should be high enough to resist unfolding from the C-terminus during translocation. Our results revealed that when adjacent to the folded RTX block IV, the mechanical stability of RTX block V is further increased. To mechanically unfold a translocated RTX block V, a much higher force (~30 pN vs ~14 pN) is required, making the unfolding more difficult under physiological conditions. Therefore, as the secretion process proceeds, the translocated RTX block V becomes more stable and prevents the unfolding initiated from the C-terminus of CyaA. As RTX block I-III will be secreted after RTX block IV, it is plausible that the folded RTX block III might also enhance the mechanical stability of block IV by the formation of another β-roll. It is interesting to note that the three expected RTX motifs at the N-terminal end of RTX block IV were unstructured, as revealed in this study. The mechanically compliant characteristics of these RTX sequences might result from the lack of folded RTX block III, which is worth exploring in the future studies. 5.5 Conclusions By combining SMFS and protein engineering, we investigated the mechanical unfolding and folding behaviors of RTX block IV of bacterial toxin CyaA, which is adjacent to RTX block V and is secreted right after RTX block V. Our results showed that RTX block IV does not fold independently, but requires a folded structure of RTX block V as a template. Our results revealed that the unfolding and folding of holo-RTX block IV mostly occur via a four-state pathway, involving the formation of two intermediate states. Our results revealed that RTX block V is more stable in the presence of a folded RTX block IV, implying that the stability of folded RTX block V is further enhanced as the secretion proceeds. Our studies provide detailed biophysical 155 information associated with RTX domains, which could open up new potential avenues of developing therapeutics to combat Bordetella pertussis. 5.6 Experimental Section 5.6.1 Protein Engineering. The gene encoding RTX block IV with restriction sites (5’ BamHI and 3’ BglII and KpnI) was custom synthesized (GeneScript). RTX block IV gene was first subcloned into the pUC19 vector via BamHI and KpnI restriction sites. Then the gene encoding RTX block V263 with restriction sites (5’ BamHI and 3’ BglII and KpnI) was inserted into the construct pUC19/RTX block IV plasmid by alternating enzyme digestion and ligation steps to construct pUC19/RTX block IV-RS-V, where RS are arginine and serine resides generated by the reconstitution between the BglII and BamHI restriction sites. The additional amino acid residues RS were then removed to obtain the construct pUC19/RTX block IV-V using megaprimer approach of site-directed mutagenesis.233 Following our well-established iterative molecular biology strategy,231 we constructed the gene of NuG2-RTX block IV-NuG2 and NuG2-RTX block IV-V-NuG2. The genes were then subcloned into a modified expressing vector pQE80L-CC, which was constructed as described,206, 263 to build pQE80L/Cys-NuG2-RTX block IV-NuG2-Cys and pQE80L/Cys-NuG2-RTX block IV-V-NuG2-Cys, respectively. The genes encoding 6 mutants of RTX block IV-V (S1396C, T1411C, A1429C, S1447C, F1465C and T1483C) were obtained by megaprimer approach of site-directed mutagenesis. These genes were then subcloned into a modified expressing vector pQE80L-Cys, which carried a cassette allowing to add one cysteine residue only at C-terminus of the target protein, to build pQE80L/RTX block IV-V mutant-Cys. All the constructed genes were confirmed by DNA sequencing. The protein sequences of these constructs are shown in Appendix. 156 Protein expression and purification were performed using the same procedure as described in chapter 2 (2.5.1). 5.6.2 Preparation of DNA-protein Chimera. DNA handles and DNA-protein chimera were prepared using the same procedure as described in chapter 2 (2.5.2). 5.6.3 Single-Molecule OT Experiment. The single-molecule OT experiments (Figure 5.1B) were carried out in the same way as described in chapter 2 (2.5.3). 5.6.4 Measuring Force-Extension Relationship of Protein Unfolding The force-extension relationship of protein unfolding was measured in the same way as described in chapter 2 (2.5.4). 5.6.5 Calculating the Unfolding and Folding Kinetics. The unfolding and folding rate constants were calculated in the same way as described in chapter 3 (3.6.5). 157 Chapter 6: Summary and Future Work 6.1 Thesis Summary Understanding how proteins unfold/fold is a challenging problem. With the help of new experimental and computational techniques, people have gained many insights into the molecular mechanisms of protein unfolding/folding. OT is a powerful SMFS tool and has been widely employed to reveal the unfolding/folding of many proteins at the single-molecule level. In the introductory chapter, I discussed the protein folding problem and how OT was used to probe protein unfolding/folding. Additionally, I reviewed some significant advances in the field of protein unfolding/folding using OT. In the following chapters, I used OT to study two important classes of proteins, namely knotted/slipknotted proteins and Ca2+-binding proteins. In chapter 2, OT and SMD simulations were combined to investigate the mechanical unfolding and folding of the slipknotted protein PADC. Upon stretching from the N- and C-termini, PADC can be mechanically unfolded and untied into an unstructured polypeptide. Both two-state and three-state unfolding pathways were observed. The unfolding intermediate state was not well defined but showed a range of structures. The SMD simulations demonstrated the mechanical untying process of PADC, revealing that the friction of polypeptide sliding may lead to the formation of many intermediate states in the unfolding of PADC. Upon relaxation, a small portion of the unfolded PADC can fold and regain the slipknotted conformation, while a large percentage was observed to misfold, resulting in the complex folding behavior of PADC. Our results revealed the complexity of the unfolding and folding mechanism of such a complex slipknotted protein. In chapter 3, a trefoil knotted protein TrmD was mechanically tightened, untied and retied at the single-molecule level. Our results showed that upon stretching from the N- and C-termini, 158 TrmD can be mechanically unfolded into an unstructured polypeptide with a tightened trefoil knot. A compaction process of the trefoil knot was observed when TrmD unfolded at low forces. The mechanical unfolding was observed to occur via bifurcated pathways, including the two-state and three-state pathways. Upon relaxation, the unfolded and knotted TrmD can rapidly fold back into its native state. By using a truncated TrmD variant, we unfolded and untied the trefoil knot. Our results showed that the folding of TrmD from the unfolded polypeptide chain without the preformed knot is significantly slower, suggesting that knotting was the rate-limiting step during the folding of the whole knotted protein. Our studies provided many mechanistic insights into the unfolding/folding mechanism of trefoil knotted proteins. In chapter 4, the mechanical unfolding and folding behaviors of a Ca2+-binding protein, RTX block V, were studied by OT. Our results showed that apo-RTX block V was random coiled and did not display any mechanical resistance. Upon binding of Ca2+ ions, the unfolded polypeptide can fold into a mechanically stable structure. The mechanical unfolding and folding pathways of RTX block V were both bifurcated, including two-state and three-state pathways. By combining protein engineering and OT, we obtained a detailed picture of the unfolding/folding mechanism of RTX block V. Our results also elucidated the translocation mechanism of RTX block V and suggested that its mechanical design can facilitate the translocation through the cell membrane. In chapter 5, our results suggested that the translocation of RTX block IV occurs via a similar mechanism as that of RTX block V. Our results also showed that RTX block IV does not fold alone but requires a folded RTX block V as a template. This study also revealed the interplay between two adjacent RTX domains, which is important to enhance the stability of the translocated RTX block V. 159 Overall, this thesis demonstrates the unique capability and versatility of OT to probe protein unfolding/folding at the single-molecule level. Our findings expand the knowledge relating to unfolding/folding of knotted/slipknotted proteins and Ca2+-binding proteins, and set the stage for further investigations of these proteins. 6.2 Future Work and Outlook Protein folding is a complex physiological process. Our studies have provided mechanistic insights into the unfolding/folding of some proteins, but many aspects are still poorly understood. Further investigations are still required. In chapter 2, we combined SMFS and SMD simulations to reveal the complex unfolding mechanism of a slipknotted protein with a long threaded loop. In the SMD simulations, due to the long threaded loop, the unfolding of PADC was accompanied by a contraction of the knotting loop, leading to a somewhat tightened slipknot. However, this tightened slipknot did not persist and was eventually unfolded. Our OT studies also failed to observe this configuration. To observe a tightened slipknot caused by jamming among polypeptide chains, a slipknotted protein with a much longer threaded loop could be investigated at the single-molecule level. Thymidine kinase, a slipknotted protein with a much longer threaded loop (188 aa residues), is a good candidate for such a purpose. Sulkowska et al. revealed the existence of a tightened slipknot conformation in the coarse grained MD simulations of thymidine kinase (Figure 6.1).205 Whether this tightened slipknot can be observed in SMFS remains unknown and is worthy of further investigations. By investigating the unfolding of slipknotted proteins with different threaded loop lengths at the single-molecule level, we could gain more knowledge relating to the unfolding mechanism of slipknotted proteins. 160 Figure 6.1 Schematics of a tightened slipknot configuration of thymidine kinase. Image adapted with permission from American Physical Society, Copyright © 2009.205 After the unfolding of PADC, we relaxed the unfolded polypeptide to test whether PADC can fold into its native state. Our results revealed that unfolded PADC can misfold with a high probability. Whether the slipknotted conformation formed correctly leaves an open question. Considering the fact that the unfolding forces of native and misfolded proteins measured in OT experiments differ a lot, it would be interesting to investigate whether the slipknot is the key for maintaining the protein stability. Proline isomerization during the unfolding/folding process might be a possible reason for misfolding of PADC. One future direction is to mutate the proline residues that might undergo isomerization to other amino acid residues, e.g., glycine, to examine whether proline isomerization is responsible for protein misfolding. The folding of knotted/slipknotted proteins in vivo should be fast and robust so that their biological functions can be properly performed. However, in chapter 2 and 3, our results showed that a large percentage of PADC molecules underwent a misfolding process, and that the folding of TrmD was significantly slower without a pre-formed knot conformation. These findings leave the question of whether the folding mechanism of knotted/slipknotted proteins in vivo is different. Previous studies have suggested that chaperone-assisted folding or cotranslational folding on the 161 ribosome can facilitate the formation of knotted structures.202, 204, 217 It will be interesting to test whether chaperones or ribosomes also help the folding of PADC and TrmD. Inspired by the pioneering work done by Kaiser and his coworkers,79, 179 the combination of OT and cell-free protein synthesis could allow us to probe whether the knotted/slipknotted conformation is formed during the translation process. For example, if the knotting process of TrmD occurs on the ribosome during protein synthesis, stretching the ribosome-nascent chain complexes could result in a similar unfolding pattern with that of wt TrmD, such as knot compaction. Similarly, this system could also be used to study the folding mechanism of slipknotted proteins, e.g., whether the folding of PADC occurs via an intermediate state that has a knotted structure. In chapter 4 and 5, we revealed that the two RTX domains at the C-terminus of CyaA shared similar mechanical properties, which ensure the efficient translocation through the cell membrane. Although we have provided many mechanistic insights into the two RTX domains and have proposed a possible translocation mechanism, the translocation of the other RTX domains still remains to be fully elucidated. More investigations are needed to study the mechanical properties of the other three RTX blocks, from I to III, to further corroborate the proposed translocation mechanism. To elucidate the translocation mechanism of the whole CyaA, we also need to use OT to study the N-terminus of CyaA, including the adenylate cyclase domain and the hydrophobic domain,111 which might display different mechanical behaviors. CyaA, as a virulent protein toxin, can create pores in the cell membrane and is responsible for whooping cough. Thus, it is important to develop therapeutics to combat these toxin-caused diseases. Our investigations on the mechanical properties and translocation pathway of the RTX domains may open up new ways to develop new drugs targeting this protein toxin. To ablate the toxic activities of CyaA, one possible strategy is to impede the secretion process of CyaA, so that 162 the toxin cannot be correctly folded and attack the host cells. Our results revealed that the folding of RTX block IV requires the folded RTX block V. 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J Mol Biol 2020, 432 (20), 5696-5710. 178 Appendix Amino acid sequences of the proteins NuG2 MDTYKLVIVLNGTTFTYTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE PADC MNAEINPLHAYFKLPNTVSLVAGSSEGETPLNAFDGALLNAGIGNVALIRISAIMPPEAEIVPLPKLPMGALVPTAYGYIISDVPGETISAAISVAIPKDKSLSGLIMEYEGKSSKKEAEKTVREMAKIGFEMRGWELDRIESIAVEHTVEKLGAAFAAAALWYK TrmD9,246 MWIGIISLFPEMCRAITDYGVTGRAVKNGLLSIQSWSPRDFTHDRHRTVDDRPYGGGPGMLMMVQPLRDAIHAAKAAAGEGAKVIYLSPQGRKLDQAGVSELATNQKLILVSGRYEGIDERVIQTEIDEEWSIGDYVLSGGELPAMTLIDSVSRFIPGVLGHEASATEDSFAEGLLDSPHYTRPEVLEGMEVPPVLLSGNHAEIRRWRLKQSLGRTWLRRPELLENLALTEEQARLLAEFKTEHAQ TrmD Δhelix9,161: MWIGIISLFPEMCRAITDYGVTGRAVKNGLLSIQSWSPRDFTHDRHRTVDDRPYGGGPGMLMMVQPLRDAIHAAKAAAGEGAKVIYLSPQGRKLDQAGVSELATNQKLILVSGRYEGIDERVIQTEIDEEWSIGDYVLSGGELPAMTLIDSVSRFIPGVLG TrmD45,128: MWIGIISLFPEMFRAITDYGVTGRAVKNGLLSIQSWSPRDFTHDCHRTVDDRPYGGGPGMLMMVQPLRDAIHAAKAAAGEGAKVIYLSPQGRKLDQAGVSELATNQKLILVSGRYEG179 IDERVIQTEICEEWSIGDYVLSGGELPAMTLIDSVSRFIPGVLGHEASATEDSFAEGLLDSPHYTRPEVLEGMEVPPVLLSGNHAEIRRWRLKQSLGRTWLRRPELLENLALTEEQARLLAEFKTEHAQ TrmD Δhelix45,128: MWIGIISLFPEMFRAITDYGVTGRAVKNGLLSIQSWSPRDFTHDCHRTVDDRPYGGGPGMLMMVQPLRDAIHAAKAAAGEGAKVIYLSPQGRKLDQAGVSELATNQKLILVSGRYEGIDERVIQTEICEEWSIGDYVLSGGELPAMTLIDSVSRFIPGVLG RTX block V GSARDDVLIGDAGANVLNGLAGNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAMAQYPDP RTX Δhelix1529,1664 GSARDDVLIGDAGANVLNGLAGNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVD RTX block V-L5 GSARDDVLIGDAGANVLNGLAGNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRINAGADQLWFARQGNDLEIRILGGGGLGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAMAQYPDP RTX block V A1615C GSARDDVLIGDAGANVLNGLAGNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRINCGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAMAQYPDP 180 RTX block V W1645G-Y1646G GSARDDVLIGDAGANVLNGLAGNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRINAGADQLWFARQGNDLEIRILGTDDALTVHDGGRDADHRVEIIHAANQAVDQAGIEKLVEAMAQYPDP RTX block IV GAGRVDKLGEAGSSAYDTVSGIENVVGTELADRITGDAQANVLRGAGGADVLAGGEGDDVLLGGDGDDQLSGDAGRDRLYGEAGDDWFFQDAANAGNLLDGGDGRDTVDFSGPGRGLDAGAKGVFLSLGKGFASLMDEPETSNVLRNIENAV RTX block IV S1396C GAGRVDKLGEAGSSAYDTVCGIENVVGTELADRITGDAQANVLRGAGGADVLAGGEGDDVLLGGDGDDQLSGDAGRDRLYGEAGDDWFFQDAANAGNLLDGGDGRDTVDFSGPGRGLDAGAKGVFLSLGKGFASLMDEPETSNVLRNIENAV RTX block IV T1411C GAGRVDKLGEAGSSAYDTVSGIENVVGTELADRICGDAQANVLRGAGGADVLAGGEGDDVLLGGDGDDQLSGDAGRDRLYGEAGDDWFFQDAANAGNLLDGGDGRDTVDFSGPGRGLDAGAKGVFLSLGKGFASLMDEPETSNVLRNIENAV RTX block IV A1429C GAGRVDKLGEAGSSAYDTVSGIENVVGTELADRITGDAQANVLRGAGGADVLCGGEGDDVLLGGDGDDQLSGDAGRDRLYGEAGDDWFFQDAANAGNLLDGGDGRDTVDFSGPGRGLDAGAKGVFLSLGKGFASLMDEPETSNVLRNIENAV RTX block IV S1447C 181 GAGRVDKLGEAGSSAYDTVSGIENVVGTELADRITGDAQANVLRGAGGADVLAGGEGDDVLLGGDGDDQLCGDAGRDRLYGEAGDDWFFQDAANAGNLLDGGDGRDTVDFSGPGRGLDAGAKGVFLSLGKGFASLMDEPETSNVLRNIENAV RTX block IV F1465C GAGRVDKLGEAGSSAYDTVSGIENVVGTELADRITGDAQANVLRGAGGADVLAGGEGDDVLLGGDGDDQLSGDAGRDRLYGEAGDDWFCQDAANAGNLLDGGDGRDTVDFSGPGRGLDAGAKGVFLSLGKGFASLMDEPETSNVLRNIENAV RTX block IV T1483C GAGRVDKLGEAGSSAYDTVSGIENVVGTELADRITGDAQANVLRGAGGADVLAGGEGDDVLLGGDGDDQLSGDAGRDRLYGEAGDDWFFQDAANAGNLLDGGDGRDCVDFSGPGRGLDAGAKGVFLSLGKGFASLMDEPETSNVLRNIENAV The sequences of primers for preparing dsDNA handles dsDNA handle 1 (802 bp): Forward primer: 5’-Bio-CAA-AAA-ACC-CCT-CAA-GAC-CC Reverse primer: 5’-NH2-CGA-CGA-TAA-ACG-TAA-GGA-CAT-C dsDNA handle 2 (558 bp): Forward primer: 5’-Dig-CAA-AAA-ACC-CCT-CAA-GAC-CC Reverse primer: 5’-NH2-GCT-ACC-GTA-ATT-GAG-ACC-AC
Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Li, Hongbin
Investigating force-induced unfolding and folding of proteins at the single molecule level
Text