UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Characterization of interactions of TonB and Colicin M with FhuA reconstituted into Nanodiscs Mills, Allan 2013

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2013_fall_mills_allan.pdf [ 3.62MB ]
Metadata
JSON: 24-1.0165589.json
JSON-LD: 24-1.0165589-ld.json
RDF/XML (Pretty): 24-1.0165589-rdf.xml
RDF/JSON: 24-1.0165589-rdf.json
Turtle: 24-1.0165589-turtle.txt
N-Triples: 24-1.0165589-rdf-ntriples.txt
Original Record: 24-1.0165589-source.json
Full Text
24-1.0165589-fulltext.txt
Citation
24-1.0165589.ris

Full Text

Characterization of interactions of TonB and Colicin M with FhuA reconstituted into NanodiscsbyAllan MillsB.Sc., Vancouver Island University, 2010A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCEinTHE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES(Biochemistry and Molecular Biology)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)October, 2013? Allan Mills, 2013Abstract TonB-dependent transporters are ?-barrel outer membrane proteins that depend on interactions with the inner membrane protein TonB to drive import of scarce nutrients. Upon becoming ligand-loaded, TonB-dependent transporters bind TonB through a ?-strand exchange. FhuA is the TonB-dependent transporter that transports hydroxamate iron siderophores, such as ferrichrome and ferricrocin, into the periplasm and also acts as the receptor and transporter of the antimicrobial protein colicin M. The interactions of FhuA with TonB have previously been investigated in detergents, which can affect the conformations of TonB-dependent transporters and alter their interaction with TonB. To exclude the potential negative effects of detergent, FhuA was reconstituted into Nanodiscs that reconstitute a membrane-like environment suitable for biochemical analysis. Binding of TonB to FhuA was found to be strongly dependent on the ligand-loaded state of FhuA.  The binding affinity was relatively high (~200 nM) and enthalpy driven, suggesting a disorder-to-order interaction occurring during the ?-strand exchange. Colicin M also bound Nanodisc reconstituted FhuA with a high affinity (~3.5 nM) through an entropy driven interaction that may reflect a hydrophobic interaction. The ligand ferricrocin inhibited the binding of colicin M to FhuA. While TonB is required for transport of colicin M into cells, colicin M was not observed to cause the recruitment of TonB to FhuA. Finally, the conformation of FhuA was investigated in the presence of ferricrocin and colicin M by partial proteolysis.  iiPreface The following publication was used as a basis for text and figures shown in this thesis. The contributions of supporting authors are also described.   Mills, A., Le, H-T., Coulton, J.W., and F. Duong. 2013. Evaluating the FhuA interactions in a detergent-free nanodisc environment. Submitted. I wrote the first draft that was then edited by my supervisor. All experiments were performed by me. All figures were designed and produced by me. Dr. Hai-Tuong Le created and provided the membrane scaffold protein truncation mutant L156 for experiments. Dr. James W. Coulton provided the FhuA expressing plasmid pHX405 and the E. coli strain AW740. Dr. Coulton also provided the iron siderophore ferricrocin used in this study. Initial reconstitution experiments on FhuA were performed by Jean-Fran?ois Montariol and cloning of the truncated TonB fragment was performed with the assistance of Jean-Fran?ois Montariol and Xiao Xiao Zhang. iiiTable of contentsAbstract..........................................................................................................................................iiPreface...........................................................................................................................................iiiTable of contents...........................................................................................................................ivList of tables.................................................................................................................................viiList of figures..............................................................................................................................viiiList of equations............................................................................................................................ixList of abbreviations......................................................................................................................xAcknowledgements.......................................................................................................................xiDedication.....................................................................................................................................xii1. Introduction................................................................................................................................1 1.1 TonB-dependent transporters....................................................................................11.2 TonB..............................................................................................................................21.3 TonB transport models................................................................................................51.4 FhuA..............................................................................................................................71.5 FhuA structure.............................................................................................................91.6 FhuA signaling and transport...................................................................................121.7 Colicin M.....................................................................................................................141.8 Colicin M structure....................................................................................................151.9 Transport of Colicin M..............................................................................................181.10 Nanodiscs..................................................................................................................20iv1.11 Objectives..................................................................................................................222. Materials and methods............................................................................................................24 2.1 Materials......................................................................................................................24 2.2 Cloning of TonB..........................................................................................................24  2.2.1 Polymerase chain reaction conditions......................................................24  2.2.2 Site-directed mutagenesis..........................................................................26 2.3 TonB expression.........................................................................................................27 2.4 FhuA expression.........................................................................................................28 2.5 Colicin M expression..................................................................................................29 2.6 Nanodisc reconstitution.............................................................................................30 2.7 Isothermal titration calorimetry...............................................................................31 2.8 Analytical gel filtration..............................................................................................33 2.9 Multiangle light scattering........................................................................................33 2.10 Trypsinization of Nanodisc FhuA...........................................................................33 2.11 Other methods..........................................................................................................343 Results........................................................................................................................................35 3.1 Incorporation of FhuA into Nanodiscs.....................................................................35 3.2 Siderophore enhances recruitment of TonB to Nanodisc FhuA.............................373.3 Thermodynamics of Nanodisc FhuA and TonB complex........................................393.4 Nanodisc FhuA binds TonB in a 1:1 molar ratio......................................................413.5 Colicin M binding to Nanodisc FhuA is inhibited by ligand...................................453.6 Thermodynamics of Colicin M binding to Nanodisc FhuA....................................45v3.7 Colicin M does not promote binding of TonB to FhuA...........................................473.8 Conformational changes in FhuA caused by ligand and Colicin M.......................504 Discussion..................................................................................................................................55 4.1 Reconstitution of FhuA into Nanodiscs....................................................................55 4.2 Recruitment of TonB to Nanodisc FhuA..................................................................56 4.3 The stoichiometry between Nanodisc FhuA and TonB...........................................574.4 Colicin M interactions with Nanodisc FhuA............................................................584.5 Ternary complex of FhuA, TonB, and Colicin M....................................................605 Conclusions................................................................................................................................626 Future directions.......................................................................................................................63References.....................................................................................................................................64viList of tables Table 2.1: Polymerase chain reaction program...............................................................25 Table 2.2: Mutagenic polymerase chain reaction program.............................................27 Table 2.3: Primers...........................................................................................................27 Table 3.1: Isothermal titration calorimetry results.........................................................50viiList of figures Figure 1-1: Crystal structures of TonB..............................................................................4Figure 1-2: Competing models of TonB mediated transport.............................................7Figure 1-3: Crystal structure of FhuA...............................................................................9 Figure 1-4: Structure and topology of the TonB transport system..................................14Figure 1-5: Crystal structure of Colicin M......................................................................18 Figure 1-6: Hypothesized model of Colicin M transport................................................19 Figure 1-7: Overview of Nanodisc reconstitution...........................................................21 Figure 3-1. Reconstitution of FhuA into Nanodiscs........................................................36 Figure 3-2. Formation of a complex of Nd-FhuA-TonB32-239......................................39 Figure 3-3. Binding affinity between Nd-FhuA and TonB32-239...................................41 Figure 3-4. Binding stoichiometry between Nd-FhuA and TonB32-239.........................44 Figure 3-5: Ferricrocin prevents binding of Colicin M to Nd-FhuA...............................47 Figure 3-6: Colicin M does not seem to trigger the binding of TonB to Nd-FhuA.........49Figure 3-7: Colicin M and Ferricrocin affect the conformation of Nanodisc reconstituted FhuA................................................................................................................................53viiiList of equationsEquation 2-1: Equilibrium association constant..........................................................................32ixList of abbreviations ABC  ATP Binding CassetteATP  Adenosine TriphosphateDDM   n-Dodecyl-?-D-maltopyranosideDM   n-Decyl-?-D-maltopyranosideIPTG  Isopropyl-?-D-thiogalactopyranosidekDa  kilodalton = 1000 grams/moleLB  Luria-Bertani brothLDAO  Lauryldimethylamine oxideMSP  Membrane Scaffold ProteinNd-FhuA Nanodisc FhuAOctyl-POE n-Octyl-Polyoxyethylene PAGE  Poly-acrylamide gel electrophoresisPCR  Polymerase Chain ReactionPDB  Protein Data BankPMSF  Phenylmethylsulfonyl fluorideSDS  Sodium Dodecyl Sulfate xAcknowledgements I would first like to thank my supervisor, Dr. Franck Duong, for providing me with this fascinating project to research. I would also like to thank my advisory committee and external examiner, Dr. Robert Hancock, Dr. Joerg Gsponer, and Dr. Calvin Yip, for their support and recommendations. Many members of the lab have also been incredibly supportive and helpful during this project including H. Bao, S. MacDonald, S.-H. Choi, C. Chan, X. Zhang, M. Carlson, J.-F. Montariol, H. Li, K. Dalal, and many more. Lastly, I want to thank my friends and family for supporting me during these past years. The work presented in this thesis was generously funded through fellowships provided by the Natural Sciences and Engineering Program of Canada and the University of British Columbia. xiDedicationTo my familyxii1. Introduction1.1 TonB-dependent transporters Gram-negative bacteria, such as Escherichia coli, have a dual membrane organization, an inner and outer membrane, that complicates the process of transport. Nutrients in the space between membranes, termed the periplasm, are transported across the inner membrane to the cytoplasm by highly specialized transporters (Davidson and Chen, 2004). Transport across the outer membrane also requires different type of transporters. Facilitated diffusion occurs by porin channel proteins. These porins are ?-barrel proteins with a central lumen that permits passage from the extracellular space to the periplasm. In the case of LamB, for example, a ?greasy slide? mechanism composed of several aromatic residues facilitates the passage of maltose into the periplasm (Nikaido, 2003). Facilitated diffusion allows the transport of small molecules following their concentration gradient. This mechanism is insufficient for the transport of scarce and large nutrients. Gram-negative bacteria have evolved specialized transporters for large (over 600 Da) and scarce nutrients (Nikaido, 2003). Active transport is generally accomplished using TonB-dependent transporters, a group of related proteins with a conserved structure. Sequence alignment has shown that TonB-dependent transporters have 20% or greater homology (Ferguson and Deisenhofer, 2004; Krewulak and Vogel, 2008). Several TonB dependent transporters, such as FhuA (Ferguson et al. 1998; Locher et al. 1998), BtuB (Chimento et al. 2003), FpvA (Cobessi et al. 2005), FecA (Ferguson et al. 2002), and FepA (Buchanan et al. 1999), have been crystallized: all of these transporters are 22-stranded ?-barrels composed of approximately 600 C-terminal residues with approximately 150 N-terminal residues forming a globular plug domain that occludes the channel of the ?-barrel (Ferguson and Deisenhofer, 2002; Chakraborty et al. 12006 and references therein). These larger ?-barrels are thought to permit the import of larger ligands that are bound with high affinity to binding pockets on the extracellular face of the transporters. A conserved motif near the extreme N-terminus, termed the ?TonB box?, acts as an interacting site for the C-terminus of the inner membrane protein TonB (Howard et al. 2001; Ferguson and Deisenhofer, 2002).1.2 TonB  TonB is a 239 amino-acid transmembrane protein located in the inner membrane that has a role in coupling an energized membrane state with transport (Hancock and Braun, 1976; Moeck and Coulton, 1998). TonB has three domains: transmembrane domain of residues 1-32, a central periplasm spanning domain of residues 33-100, and a C-terminal domain of residues 103-239 (Chimento et al. 2005). The transmembrane domain of TonB interacts with two inner membrane proteins: ExbB and ExbD. ExbB and ExbD are homologs of the flagellar motor proteins MotA and MotB that use the proton motive force (PMF) (Cascales et al. 2001). It is thought that the TonB-ExbB-ExbD complex interacts with ligand-loaded transporters and utilizes the PMF as an energy source to power the transport of bound nutrient ligands across the outer membrane (reviewed in Noinaj et al. 2010). Mutations in the transmembrane domain of TonB at the position His20 abolish TonB transport activity. His20 is located at the interface with ExbB. (Larsen and Postle, 2001).  TonB has a rigid, elongated structure that allows it to span the periplasm. This characteristic of TonB is provided by the proline-rich motif of residues 66-100 which is composed of a number of Pro-Glu and Pro-Lys repeats. The rigidity provided by this motif is 2thought to allow the central domain of TonB to extend ~100? to span the periplasm (K?hler et al. 2010). Deletion of the proline-rich motif does not abrogate transport and, therefore, contributes primarily to allowing TonB to reach the outer membrane (Larsen et al. 1993). TonB constructs of residues 155 to 239 form dimers in solution  (Chang et al. 2001). The dimerization interface involves the ?4 strands at the extreme C-terminus of the protein (Figure 1-1A). The C-terminal 92 residues of TonB also form a dimer; however, the structure of this TonB fragment was radically different as the binding interface between protomers is much smaller (K?edding et al. 2005) (Figure 1-1B). Deletion of the proline-rich motif leads to monomerization, suggesting a possible role of the proline-rich motif in dimer formation (Khursigara et al. 2005a). The notion that TonB is functional as a dimer remain controversial. The C-terminus of TonB and the C-terminus of TolA from Pseudomonas aeruginosa have significant homology, although there are no evidence for dimerization of TolA (Peacock et al. 2005). Another TonB homologue, TonB2, from Vibrio anguillarum is also monomeric. The ?4 strand (residues 235-239) is a primary contributor to dimerization, and this structure is absent in TonB2 (L?pez et al. 2009). The ?4 strand forms the major interacting interface between TonB protomers and its absence may affect dimerization (Chang et al. 2001; K?edding et al. 2005). Certain types of detergents used in interaction studies have an effect on the oligomerization of TonB and may elicit formation of a dimer (Moeck and Lettelier, 2001; Khursigara et al. 2004).  The C-terminal domain of TonB interacts with the TonB box. The TonB box, not visible in crystal structures due to the intrinsically unstructured nature (Locher et al. 1998; Ferguson et al. 1998), becomes visible in co-crystal of TonB with the transporters FhuA and BtuB (Pawelek et al. 2006; Shultis et al. 2006). ?-strand exchange occurs between the TonB box of transporters 3and TonB causing a transition from disorder to ordered structure (Brillet et al. 2007) (Figure 1-1 C, D). Figure 1-1: Crystal structures of TonB.(A) Crystal structure of TonB C-terminal fragment representing amino-acid residues 164-239 revealing a highly intertwined dimeric structure (PDB: 1IHR). (B) Structure of a larger TonB dimeric fragment representing residues 148-239 showing a much smaller ?-strand interface (PDB: 1U07). (C) Crystal structure of residues 8-24 of the FhuA N-terminus (orange), including the TonB-box, in complex with residues 158-235 of the TonB C-terminus (blue). The interaction shows the ordered structure of the TonB box resulting from ?-strand exchange between FhuA and TonB (PDB: 2GRX). (D) Structure of residues 5-24 of the N-terminus of the TonB-dependent cyanocobalamin transporter BtuB (orange) in complex with the C-terminal fragment of TonB (residues 153-233) (blue) showing the ?-strand exchange between the TonB box and TonB (PDB: 2GSK).41.3 TonB transport models The mechanism of action of TonB has not been elucidated; but, several models have been proposed: One model is the ?shuttle hypothesis?, proposed because TonB is found associated with the outer membrane as well as the inner membrane (Letain and Postle, 1997). This model posits that TonB in an unenergized state is associated with the inner membrane, where it can interact with ExbB and ExbD. Upon becoming energized TonB can disengage itself from the inner membrane and become associated with transporters in the outer membrane to drive transport (Postle and Kadner, 2003). However, a fusion of TonB with the cytoplasmic domain of ToxR from Vibrio cholerae maintains activity  (Gresock et al. 2011). TonB is sufficiently long to span the periplasm and does not need to shuttle off the inner membrane (K?hler et al. 2010) (Figure 1-2B).  Another model is the ?propeller model?, in which TonB forms a dimer and undergoes rotary motion to displace or unfold the plug domain of the transporter to permit import of the ligand. This model is based on the homology of ExbB and ExbD to the flagellar motor proteins MotA and MotB, which also require the PMF for activity (Cascales et al. 2001). There continues to be debate about the stoichiometry of TonB in complex with transporters  (Chang et al. 2001; Sauter et al. 2003). While TonB has been observed as a dimer with the ligand-loaded FhuA (Khursigara et al. 2004; 2005b), it has also been observed as a monomer with BtuB (Freed et al. 2013). Dimerization may not be a necessary aspect of TonB-mediated transport because homologues of TonB that do not dimerize still mediate transport, assuming the mechanism of transport is conserved (L?pez et al. 2009; Krewulak and Vogel, 2010) (Figure 1-2C).  5 Another model is the ?pulling model?. TonB may undergo conformational changes that apply force perpendicular to the membrane. Water hydration of the plug domain is believed to decrease the activation energy required to induce unfolding by providing lubrication (Feraldo-G?mez et al. 2003). The conserved residues Arg166 of TonB and Glu56 of the plug domain of FhuA form an electrostatic interaction where a force can be applied perpendicular to the plug (Pawelek et al. 2006). A conformational change in TonB could pull on this residue allowing the unfolding of the plug domain to permit passage of the bound ligand (reviewed in Krewulak and Vogel, 2010) (Figure 1-2D).   The ?scaffold model? proposes that a periplasmic binding protein (PBP), such as FhuD or BtuF, binds to TonB (Carter et al. 2006; James et al. 2009). Binding of the PBPs to TonB is thought to orient the PBP directly below the periplasmic face of the transporter allowing it to bind the ligand after the plug domain has been dislocated or unfolded. This model was substantiated by phage display, dynamic light scattering, fluorescence spectroscopy, and surface plasmon resonance on interactions between TonB and the PBPs FhuD and BtuF (Carter et al. 2006; James et al. 2009). The PBP will dissociate from TonB after binding ligand and can then present this substrate to the corresponding ABC transporters for transport across the inner membrane. This model does not demonstrate the mechanism of energy transfer or plug domain movement during transport; however, this model does show a possible arrangement of all protein components of transport to maximize efficiency (reviewed in Krewulak and Vogel, 2010) (Figure 1-2E). 6Figure 1-2: Competing models of TonB mediated transport.(A) Pre-interaction state of the TonB-dependent transporter FhuA (orange) and TonB (blue) shown in the outer and inner membrane, respectively. ExbB (magenta) and ExbD (purple) are also shown in the inner membrane. (B) The ?shuttle? model: TonB disengages from the inner membrane to interact with FhuA in the outer membrane to mediate transport. (C) The ?propeller? model: a dimer of TonB binds the TonB box of the transporter and undergoes rotary motion to drive the transport of ligand into the periplasm. (D) The ?pulling? model: TonB undergoes a conformational change to pull the TonB box perpendicularly to the membrane. (E) The ?PBP-assisted? model: the periplasmic binding protein (PBP) FhuD (yellow) binds to TonB and is localized at the periplasmic face of FhuA to immediately bind ligands after transport.1.4 FhuA FhuA was initially discovered through a screen of Escherichia coli strains with mutations that rendered them virus resistant. Two mutants with T1 bacteriophage resistance were labeled tonA and tonB (for T-one A and B) (reviewed in Braun, 2009). TonA would later be renamed as FhuA (Ferric hydroxamate uptake A) to reflect that iron siderophores are transported by this protein (Kadner et al. 1980). 7 FhuA is the transporter of iron hydroxamate siderophores, such as ferrichrome and ferricrocin, and acts as the receptor for bacteriophages T1, T5, and ?80, as well as antibiotic compounds and proteins albomycin, rifamycin CGP 4832, colicin M and microcin J25 (Braun et al. 1973; Stefanska et al. 2000; Ferguson et al. 2001b; reviewed by Braun, 2009). FhuA, like other TonB-dependent transporters, has a 553 residue 22-stranded C-terminal ?-barrel and an N-terminal 160 residue globular plug domain (Locher et al. 1998; Ferguson et al. 1998) (Figure 1-3A, B). FhuA binds iron hydroxamate siderophores at a binding pocket on the extracellular face of the protein. This binding site is composed of both hydrophilic and hydrophobic aromatic residues from both the plug domain and the extracellular loops. The binding of ligand in the pocket likely uses the aromatic residues to orient the siderophore with the complexed iron atom facing in toward the periplasm with the opposite end of the molecule exposed to the extracellular solvent. This orientation of the siderophore then allows the formation of hydrogen bonds between the siderophore and other residues lining the binding pocket, which explain the high-affinity binding of siderophore by FhuA (Locher et al. 1998). 8Figure 1-3: Crystal structure of FhuA.(A) Crystal structure of FhuA oriented as it would be in the lipid bilayer showing the primarily ?-barrel structure. Surface exposed loops on the extracellular side (top) and periplasmic face (bottom). The ?-barrel (orange) is shown with residues 621-714 removed to show the plug domain (blue). The TonB box of FhuA is not visible due to the disorder present in the TonB box. (B) FhuA crystal structure viewed from the extracellular face along the barrel axis. The globular plug domain (blue) is shown occluding the lumen of the ?-barrel (PDB: 1BY3).1.5 FhuA structure  The TonB box of FhuA (residues 7-DTITVTA-13) has not been shown in crystal structures of either the apoprotein or ligand loaded state of FhuA because the TonB box is an intrinsically unfolded motif (Locher et al. 1998; Ferguson et al. 1998). The constitutively unfolded structure of the FhuA TonB box has also been demonstrated using electron paramagnetic resonance and site-directed spin-labeling (Kim et al. 2007). Comparisons between the crystal structure of ligand-loaded and apoprotein state of FhuA show that binding of the ferric 9siderophore ligand results in the unwinding of a short helix, termed the ?switch helix?, of residues 24 to 29. The unwinding of this helix causes significant movement of the N-terminus of the protein, including the TonB box, by approximately 17? from a position within the ?-barrel to a periplasm exposed position (Locher et al. 1998; Ferguson et al. 1998). It was postulated that this movement of the TonB box is important to signal the ligand-loaded state of FhuA and to recruit TonB for transport. Crosslinking of cysteine residues engineered into both the plug and barrel at position Thr27 and Pro533 such that movement of the N-terminus was prevented resulted in the abolishment of siderophore transport (Endri? et al. 2003). Recent research using site-directed spin labeling has contested this model of switch helix unwinding by showing that the helix remains unwound in both the apoprotein and ligand-loaded state. This later finding suggests that a different mechanism is necessary to communicate the ligand-loaded state of FhuA (Kim et al. 2007).  TonB binding to FhuA and the effect of the ligand has been studied in vitro. Truncated monomeric TonB (residues 33 to 239) was shown by analytical ultracentrifugation to form a dimer in the presence of ligand-loaded FhuA (Khursigara et al. 2004). This indicates that TonB interactions with the ligand-loaded state, as opposed to the apoprotein state, elicits dimerization of TonB. Surface plasmon resonance techniques showed that the ligand-loaded state of FhuA enhanced the binding of TonB to FhuA. A flexible CM4 dextran surface plasmon resonance chip that allowed immobilized TonB to recruit a second TonB into a dimer was compared to an inflexible CM1 chip that prevented dimerization.  It was found that only on the flexible chip did ligand have an enhancing effect on the recruitment of TonB by FhuA (Khursigara et al. 2005b). This result indicates that dimerization of TonB is important for recruitment to the ligand-loaded 10FhuA. Recent research into another TonB-dependent transporter, BtuB, has shown that TonB binds exclusively as a monomer to the ligand-loaded state of the transporter (Freed et al. 2013). The cocrystal structures of FhuA and BtuB in complex with TonB also show a monomer, rather than a dimer (Pawelek et al. 2006; Shultis et al. 2006). As noted earlier, the TonB box of FhuA is constitutively unfolded; however, the TonB box is observable in the cocrystal structure of FhuA and TonB in complex. A ?-strand exchange occurs between the TonB box of FhuA aligning parallel to the ?3 strand in the C-terminal region of TonB in a disorder-to-order transition. This ordered structure in complex with TonB allowed the first observation of the TonB box using crystallographic techniques (Pawelek et al. 2006; Brillet et al. 2007). Conformational changes have been shown to occur in FhuA due to the binding of siderophore ligand and TonB (James et al. 2008). Trypsinization, circular dichroism spectroscopy, and Fourier transform infrared (FTIR) spectroscopy were used to ascertain conformational changes that occur in FhuA due to the binding of siderophore ligand (Moeck et al. 1996). Crystallographic evidence for conformational changes in FhuA upon binding of ligand were confined primarily to the plug domain, very little conformational change was observed within the structure of the ?-barrel (Locher et al. 1998: Ferguson et al. 1998). However, kinetic analysis of monoclonal antibody binding to the external loops of the FhuA ?-barrel show that a conformational change occurs upon binding of both ferricrocin and TonB (James et al. 2008). This may explain the difference in the energy and TonB-dependent binding of bacteriophages T1 and ?80 to FhuA and the absence of significant structural changes in the ?-barrel in crystallographic structures is likely the result of non-physiological crystallization conditions (reviewed in Braun, 2009).111.6 FhuA signaling and transport To accommodate passage of ligands, such as the iron siderophore ferricrocin, or bactericidal proteins, such as colicin M, the plug domain of FhuA must either unfold or dislodge entirely from the barrel. Cysteine residues were engineered into both the plug and barrel domains and disulfide cross-linked to tether the plug to the interior of the barrel. Transport of radio-labelled ligand was decreased but still significant (Eisenhauer et al. 2005). BMCC biotin probing of cysteine residues of the plug and barrel domains during colicin M transport failed to show any labeling. These result show that the plug is likely unfolded rather than dislocated during transport of ferricrocin and colicin M (Braun et al. 2012). This result is at odds with the other TonB-dependent transporter FepA, which showed labeling of several cysteine residues of the plug domain during transport of colicin B into the periplasm (Devanathan and Postle, 2007). It is possible that in some instances of transport the plug domain is displaced out of the barrel of some TonB-dependent transporters while in other instances transport requires unfolding of the plug domain. Deletion of the plug domain (residues ?1-160) results in conversion of FhuA into an open channel that facilitates diffusion of several solutes. Interestingly, co-expression of constructs of FhuA ?-barrels lacking the plug domain and plug domains lacking the ?-barrel results in restored FhuA activity (Braun et al. 2003). This shows that the ?-barrel and plug domain of FhuA are assembled either in the periplasm or during insertion into the outer membrane (Braun et al. 2003; reviewed in Braun 2009).  Despite the structural similarities between FhuA and other TonB dependent transporters there are also many differences in transmembrane signaling of their ligand-loaded state. There is 12crystallographic evidence that both FhuA and FecA possess a switch helix that may unwind upon ligand binding, but no other TonB-dependent transporter employs this secondary structure (Ferguson et al. 1998; Ferguson et al. 2002). While FhuA possesses a constitutively unfolded TonB box that is unstructured regardless of the ligand-loaded state of FhuA, the TonB boxes of FecA and BtuB are folded in the apoprotein state and become disordered upon ligand binding (Kim et al. 2007). The TonB box of BtuB is also ?docked? into a position within the inside of the ?-barrel. Upon becoming ligand loaded the BtuB TonB box becomes unstructured and ?undocks? from the the interior of the ?-barrel to where it is more accessible to recruit TonB (Xu et al. 2006). Binding of TonB to FecA was found to be low affinity, regardless of the ligand-loaded state of FecA (Freed et al. 2013). It has been proposed that FecA is auto-regulated in expression by a large N-terminal extension (Peacock et al. 2006), which may block the TonB box and hinder TonB recruitment to FecA (Freed et al. 2013). 13Figure 1-4: Structure and topology of the TonB transport system. The topology of TonB and the TonB transport system is shown. ExbB is thought to have three transmembrane domains (Kampfenkel and Braun, 1993), ExbD and TonB each have only a single transmembrane domain (Postle and Skare, 1988; Kampfenkel and Braun, 1992). TonB (blue) is shown with the transmembrane domain at the N-terminus spanning the inner membrane (residues 1-32). The proline-rich region is in the central region of the TonB between residues 66-100. The C-terminal domain of TonB interacts with the TonB box of the ligand-loaded transporter FhuA (PDB: 2GRX).1.7 Colicin M Colicins are antimicrobial proteins expressed by certain strains of E. coli to kill competing strains. To enter an E. coli cell the colicin must first bind to a receptor on the outer membrane and then be transported into the cell to exert their killing activity (reviewed in 14Cascales et al. 2007; reviewed in Braun, 2009). Colicin M is a small colicin protein (approximately 29 kDa) that utilizes FhuA both as a receptor to adhere to the cell surface and as a transporter to access the periplasm (Braun et al. 2012). Colicin M is a member of the group B colicins, which usurp the function of the TonB-ExbB-ExbD transport system to access the periplasm (Cascales et al. 2007). Colicin M kills targeted cells by inhibiting the synthesis of murein by cleaving between bactoprenol and 1-pyrophospho-MurNAc-(pentapeptide)-GlcNAc (Schaller et al. 1982; Ghachi et al. 2006). 1.8 Colicin M structure Colicins share a common arrangement into three separate domains: an N-terminal transport domain, a receptor binding domain, and a C-terminal activity (cell killing) domain. Unlike larger colicins, colicin M does not have lengthy sequences of amino-acids separating the individual domains (Braun, 2009; Braun et al. 2012).  The N-terminal transport domain of colicin M (residues 1-35), like other group B colicins, has a TonB box sequence (residues 2-ETLTVHA-8) (Postle and Larsen, 2007), and mutations introduced into this TonB box result in inhibition of killing activity (Pilsl et al. 1993). Osmotic shock to allow colicin M entry into cells, thereby bypassing the necessity of active transport, results in cell killing by colicin M with mutations in the TonB box. This shows that mutations in the TonB box do not affect the cell killing activity of colicin M; but, that the TonB box is required for transport (Pilsl et al. 1993). Corresponding suppressing mutations introduced into TonB restores sensitivity of cells to this TonB-box mutant colicin M and demonstrates that colicin M interacts directly with TonB. Removal of the transport domain by proteinase K 15digestion was also shown to inhibit killing of cells by colicin M except when digested colicin M was osmotically shocked across the outer membrane, indicating the necessity of this domain for transport into cells (Dreher et al. 1985).  The central domain of colicin M, encompassing residues 36-140, is the receptor binding domain that adheres colicin M to the surface of Escherichia coli cells by binding to FhuA. Several regions of FhuA have been implicated in binding of colicin M. Synthesized hexapeptide sequences corresponding to a large loop on the extracellular face of FhuA from residues 316 to 356 were incubated with colicin M and were found to inhibit cell killing, indicating that the peptides corresponding to this extracellular loop bound to colicin M and prevented colicin M from binding to FhuA on the surface of cells (Killmann et al. 1995). Deletion of residues 21 to 128 of the plug domain of FhuA retains all FhuA activities except for colicin M sensitivity, which decreased significantly by eight-fold. This indicates that colicin M interacts with part of the plug domain upon binding to FhuA (Carmel and Coulton, 1991). A series of mutations made in colicin M identified a hydrophobic helix, termed ?1 (residues 38-VQVVYSFFQ-46), within the receptor binding domain as essential for binding to FhuA (Helbig and Braun, 2011). Helix ?1 was initially postulated to be involved in adhering colicin M to the periplasmic leaflet of the inner membrane upon accessing the periplasm (Zeth et al. 2008). When helix ?1 was deleted colicin M required a 105-fold increase in protein to inhibit cell growth to the same degree as wild-type colicin M. Colicin M with a deletion of the ?1 helix was also unable to compete with albomycin for transport into the E. coli strain Mo3, which is sensitive to albomycin but resistant to colicin M, whereas wild-type colicin M successfully competed with albomycin to inhibit cell 16killing by albomycin (Helbig and Braun, 2011). This result indicates that helix ?1, despite the high hydrophobicity of this helix, is required for binding to FhuA.  The C-terminal activity domain is responsible for exerting the toxic effects of colicin M. Interestingly, crystal structures of colicin M show that the active domain of colicin M is a mix of ? and ? structures forming an open ?-barrel structure. This active domain has structural similarity  to the membrane protein Hia from Haemophilus influenzae (Zeth et al. 2008) (Figure 1-5). Although this open ?-barrel is not considered to have direct role in the transport of the protein across the outer membrane, it is interesting the deletion or release of the last two residues of colicin M at the extreme C-terminus (residues K270 and R271) by carboxypeptidase B abolishes transport (Dreher et al. 1985; Helbig and Braun, 2011). Colicin M with deletions of the the two C-terminal residues is incapable of killing cells or inhibiting killing by competing wild-type colicin M. This result indicates that this colicin M mutant binds FhuA but is not transported and, therefore, blocks the binding site for wild-type colicin M (Dreher et al. 1985; Helbig and Braun, 2011). The precise role of these residues in transport has not yet been elucidated (Zeth et al. 2008; Helbig and Braun, 2011).17Figure 1-5: Crystal structure of Colicin M.Crystal structure of the colicin M representing residues 2-271 from N-terminus to C-terminus colored blue to red. The structure is shown in two different views at 180? from each other. Hydrophobic helix ?1 of the receptor binding domain is indicated (Zeth et al. 2008) (PDB: 2XMX).1.9 Transport of Colicin M Upon binding to FhuA, colicin M becomes trypsin sensitive, which indicates that colicin M undergoes a conformational change (Schaller et al. 1981). Transport of colicin M is proposed to involve passage through the barrel of FhuA, a conformational change from a compact state indicates that colicin M may become more linear to traverse the lumen (Zeth et al. 2008). Passage through FhuA is occluded by the plug domain of FhuA. As noted earlier, the majority of the plug domain is required for transport and it has been shown that mutations in the TonB box of FhuA that prevent FhuA from recruiting TonB also inhibit transport of colicin M (Sch?ffler and Braun, 1989; Carmel and Coulton, 1991). This has led to the model that binding of colicin M 18to FhuA promotes recruitment of TonB to the TonB box of FhuA. TonB can then displace the plug domain out of the barrel for the N-terminal transport domain of colicin M to span the channel of FhuA and enter the periplasm. The TonB box of colicin M then recruits TonB and can use the power provided by the PMF to enter the periplasm (Cascales et al. 2007; Zeth et al. 2008). In addition to the known proteins FhuA, TonB, ExbB, and ExbD (Braun et al. 1980; Fischer et al. 1989), colicin M mediated cell killing also requires FkpA, a periplasmic chaperone protein. Knockout mutations of FkpA have been shown to render cells resistant to colicin M, suggesting that the chaperone activity of FkpA is required to refold colicin M after it has been transported through FhuA (Hullmann et al. 2008) (Figure 1-6).Figure 1-6: Hypothesized model of Colicin M transport.In stage 1 colicin M binds to the FhuA in the outer membrane using the central receptor binding domain (R-domain). Binding of colicin M to FhuA induces the recruitment of TonB to the TonB box of FhuA, which then powers to movement of the plug domain in stage 2. In stage 3 the displacement of the FhuA plug domain by TonB allows the N-terminal transport domain (T-domain) into the barrel and interact directly with TonB using the TonB box of colicin M. The direct interaction of TonB with colicin M in stage 4 causes TonB to pull colicin M through the barrel of FhuA into the periplasm. In stage 5 colicin M has been refolded within the periplasm by  FkpA (not shown) where it can inhibit murein biosynthesis. 191.10 Nanodiscs Investigation of membrane proteins in vitro is often complicated by the use of detergents to solubilize membrane proteins. Detergent micelles can adversely affect the folding of different membrane proteins and also cause improper oligomerization. To properly investigate membrane proteins in vitro, membrane proteins have been reconstituted into lipid bilayers and other membrane mimetic environments, such as liposomes. Unfortunately, many interaction studies and techniques require that the membrane protein be accessible on both sides. To solve this problem nanodiscs were created using truncations of apolipoprotein A-1 to form a membrane scaffold protein (MSP) (Bayburt et al. 2002; Bayburt and Sligar, 2003; Denisov et al. 2004). The MSP forms the circumference of a nanoscale patch of lipid bilayer that can encompass a given membrane protein (Figure 1-7). These nanodiscs allow investigation of a soluble form of a given membrane protein without relying on detergents or other membrane mimetic environments (Bayburt and Sligar, 2003). Nanodiscs have been used to study cytochrome P450 (Denisov and Sligar, 2011), bacteriorhodopsin (Bayburt et al. 2006), the SecYEG translocase (Alami et al. 2007), and maltose FGK2 permease (Bao and Duong, 2012). 20Figure 1-7: Overview of Nanodisc reconstitution. FhuA solubilized from membrane and maintained in solution by detergents is incubated with the membrane scaffold protein (MSP) with detergent adsorbing biobeads that remove detergent from solution. After detergent removal by biobeads the nanodisc is subjected to size exclusion chromatography to remove both protein aggregates and MSP that has not been incorporated into nanodiscs. Detergents have been demonstrated to have adverse effects on the TonB transport system. The conformation of the TonB box of BtuB was affected by different types of detergent (Fanucci et al. 2003). FptA was shown to not bind TonB until the octyl-POE detergent was replaced with N-decyl-?-D-maltoside (DM), indicating that some detergents can adversely affect the interaction of TonB with outer membrane transporters (Choul-Li et al. 2008). Additionally, TonB oligomerization can be affected by the use of detergent. Dimerization has been observed on gel filtration when using a buffer containing lauryldimethylamine oxide (Moeck and Lettelier, 2001) and in analytical ultracentrifugation with Tween 20 (Khursigara et al. 2004).  211.11 Objectives Despite the extensive work conducted upon FhuA, TonB, and colicin M there are still unresolved issues surrounding the characterization of interactions between these proteins in a membrane mimetic environment. Previous research was performed in vitro in detergents. It has been demonstrated that the presence of detergents can affect the conformation and stoichiometry of both TonB-dependent transporters as well as truncated TonB fragments. In the work presented below, three major aspects of TonB-dependent transport were investigated:1) What is the affinity and thermodynamic parameters of TonB and colicin M interactions with the apo-protein and ligand-loaded states of FhuA?2) What is the stoichiometry of the interaction of TonB with FhuA?3) Does colicin M elicit the recruitment of TonB to FhuA in order to assemble a transport-ready complex? Does colicin M induce the same conformational changes in FhuA as ferricrocin? To address these objectives FhuA was reconstituted into nanodiscs. Native gel electrophoresis and gel filtration chromatography were employed to determine the stability of the complexes of nanodisc FhuA with TonB or colicin M. Isothermal titration calorimetry was employed to ascertain the binding affinity and thermodynamic parameters of nanodisc reconstituted FhuA interactions with TonB and colicin M in the absence or presence of the iron siderophore ferricrocin.  The stoichiometry of TonB interactions with FhuA were assessed by cysteine-crosslinking of TonB to form dimers in solution. Native PAGE analysis was used to compare 22interactions of wild-type TonB and crosslinked dimer TonB with nanodisc reconstituted FhuA to determine if wild-type TonB binds as a dimer to FhuA. To further test the stoichiometry of TonB with FhuA a multi-angle light scattering analysis was performed to determine the molecular mass of nanodisc FhuA and TonB individually as well as in complex. To test the effect of colicin M on the recruitment and stabilization of a ternary complex of nanodisc FhuA with TonB and colicin M native PAGE analysis was performed. To further evaluate the stability of the complex an analytical gel filtration chromatography assay was performed as well as isothermal titration calorimetry. A modified trypsinization technique of FhuA was used to investigate the conformational changes in FhuA due to the binding of either ferricrocin or colicin M. 232. Materials and methods:2.1 Materials: Salts and solvents were purchased from Fisher Scientific (Hampton, NH). Enzymes used for cloning were purchased from New England Biolabs, Inc, (Ipswitch, MA). The following materials were acquired as indicated: Primer oligonucleotides - Invitrogen (Carlsbad, CA). Dithiothreitol and Lauryldimethylamine-N-oxide (LDAO) - Sigma-Aldrich (St. Louis, MO). N-decyl-?-D-maltopyranoside (DDM) - Affymetrix (Santa Clara, CA). Detergent adsorbent Bio-Beads? - Bio-Rad (Hercules, CA). Purification columns and beads - GE Healthcare (Pittsburgh, PA). Phenylmethylsulfonyl fluoride (PMSF) and antibiotics - Bioshop (Burlington, ON)2.2 Cloning of TonB A plasmid expressing TonB (pBad22-TonB-His6) in BL21 strain was already available in the lab. Cloning on this plasmid was performed to clone TonB into the pET28 vector and to replace residues 1-32 of the membrane spanning region of TonB with a hexahistidine tag and a linker region. Primers used in this study are available in Table 3. The restriction enzyme sites for NdeI and XhoI, respectively, are underlined. 2.2.1 Polymerase chain reaction conditions This polymerase chain reaction condition (PCR) used approximately 0.5 ?g of template DNA, 0.3 mM dNTPs, and 1 unit of PhusionTM  DNA polymerase (NEB) incubated with recommended reaction buffer to a final reaction volume of 50 ?l in a 0.2 ml thin wall PCR 24reaction tube (Axygen). Reactions were performed in an Eppendorf PCR Mastercycler.  PCR conditions were as listed below:Table 2.1: Polymerase chain reaction program      Step Temperature Time1 98?C 2 minutes2 98?C 20 seconds3 60?C 30 seconds4 72?C 30 seconds5 Repeat step 2-4 Repeat for 20 cycles6 72?C 6 minutes7 16?C Hold PCR product was separated from template by running on a 1.0% agarose gel on 1X TBE buffer. The band corresponding to amplified PCR product was excised under low intensity UV conditions and purified using Econospin? silica centrifuge column (Epoch Life Science).  The purified TonB PCR product was then subjected to double restriction digestion by NdeI and XhoI for 2 hours at 37?C. Vector pET28a was also doubly digested with NdeI and XhoI under identical conditions. Digested PCR product and vector were then run on 1.0% agarose gel to separate the digested TonB product from terminal overhang sequences and to separate digested pET28a vector from insert. The band corresponding to the digested TonB sequence was again excised and purified as well as the digested pET28a vector and both were used in ligation.   Ligation between TonB double digested PCR product and doubly digested pET28a vector was performed at approximately 10 fold excess of TonB PCR product insert to vector. Vector and insert were incubated together with 1X Type 2 ligation buffer (New England Biolabs) and 1 unit of T4 DNA Ligase overnight at room temperature. 25 TonB-pET28 plasmid was then transformed into E. coli strain BL21 to express the truncated soluble TonB protein. Plasmid was minipreped and sequence was confirmed by sequencing by Eurofin MWG Operon.        2.2.2 Site directed mutagenesis of TonB to TonB-cys Plasmid TonB-pET28 was minipreped from BL21 expressing TonB. The total volume of the mutagenesis reaction was 25?l and was carried out in 1X HF Buffer with 1?l of template TonB-pET28 plasmid. The PCR program for mutagenesis is listed in Table 2. 2?l of PCR product was visualized on 1.0% agarose gel to confirm amplification. 10?l of PCR product was then subjected to DpnI digestion in 1X Type 4 REact buffer (Invitrogen) for 2 hours at 37?C to remove parental template plasmid. PCR product was then transformed into E. coli strain DH5?. Colonies grown on LB agar plates supplemented with kanamycin confirmed the successful transformation of DH5? with TonB-cys-pET28 plasmid. Correct insertion of the mutation was confirmed by sequencing with Genewiz Inc. Colonies of DH5? were streaked and underwent miniprep to extract the TonB-cys-pET28 plasmid for transformation and expression in E coli strain BL21. Mutagenic primers used are listed in Table 3. A glycine residue is changed to a cysteine at the codon underlined.26Table 2.2: Mutagenic polymerase chain reaction program    Step Temperature Time1 98?C 2 minutes2 98?C 30 seconds3 72?C 5 minutes4 Repeat step 2-3 Repeat for 20 cycles5 72?C 6 minutes6 16?C HoldTable 2.3: PrimersPrimer Name Sequence Vector Product*TonB NdeI 5p5?-AGTCCATATGCATCAGGTTATTGAACTACCTGCGC-3?pET28 TonB*H6*TonB XhoI 3p5?-GATCCTCGAGTTACAGAATTTCGGTGGTGCCG-3?TonB.cys.FD5?-CATCATCACAGCAGCTGCCTGGTGCCGCGCGGC-3?pET28 TonB-cysTonB.cys.FD-r5?-GCCGCGCGGCACCAGGCAGCTGCTGTGATGATG-3?2.3 TonB expression A soluble TonB fragment construct representing amino-acids 33-239 of the wild-type TonB was cloned into pET28 and transformed and expressed in the E. coli strain BL21 (DE3). This TonB contains a hexahistidine tag at the N-terminus followed by a six amino-acid linker before beginning at amino-acid 33 of the wild-type TonB. Plasmid pET28-TonB transformed into E. coli strain BL21 was grown for three hours in Luria-Bertani (LB) media supplemented with 25 ?g/ml kanamycin until OD600 reached ~0.600 and then induced with 1mM isopropyl 1-thio-?-D-galactopyranoside (IPTG). Cells were then grown for another three hours until OD600  ~1.20 then harvested by centrifugation at 6,000 rpm for 10 minutes. Cells were resuspended in buffer A (50 mM Tris-HCl, pH=7.9, 100 mM sodium chloride, 2% v/v glycerol), PMSF was added to a 27final concentration of 1 mM, and cells were lysed by three passages through a microfluidizer (Microfluidics Corp.) at 10,000 psi. Lysate was centrifuged at 55,000 rpm for 45 minutes (Beckman Type 60 Ti rotor) and supernatant was then collected. Supernatant was passed over a 1.5 mL column containing Ni2+ sepharose high performance beads equilibrated with buffer A. The column was then washed with 5 column volumes of wash buffer (Buffer A supplemented with 30 mM imidazole), then TonB was eluted with a step gradient of elution buffer (buffer A supplemented with 600 mM imidazole). TonB fractions eluted from IMAC were then subjected to strong cation exchange on a HiTrap SP FF column pre-equilibrated with buffer B (50 mM Tris-HCl, pH = 7.9, 50 mM NaCl, 2% v/v glycerol). TonB was then eluted with a 5 column volume gradient of an elution buffer of buffer A supplemented with 1M NaCl. TonB was also subjected to a final polishing step of gel filtration on a Superdex 200 HD 10/300 (Amersham) column pre-equilibrated with buffer A.  Fractions of purified TonB were analyzed by SDS-PAGE and were found to migrate at a higher molecular weight position at ~35 kDa rather than the anticipated molecular weight range of ~25 kDa based on amino-acid sequence. This is most likely because the proline-rich region of TonB retards migration on SDS-PAGE (Larsen et al. 1993).  2.4 FhuA expression Hexahistidine-tagged FhuA.H6 (hereafter referred to as FhuA) was expressed in strain AW740 (?ompF ?ompC) (Moeck et al. 1996) cells with the plasmid pHX405 (strain and plasmid donated by Dr. James Coulton of McGill University). FhuA expressing cells were grown for 16 hours in M9 minimal media supplemented with 80 ?g/ml ampicillin. Cells were harvested 28into buffer A, PMSF was added to a final concentration of 1 mM, and cells were lysed by three passes through a microfluidizer. Cell lysate was centrifuged at 6,000 rpm for 10 minutes (J25.50 rotor on a Beckman-Coulter J-E centrifuge) to remove cell debris, unbroken cells, and large inclusion bodies. The supernatant was then collected and spun at 55,000 rpm for 45 minutes. Collected pellets were resuspended in buffer A to 3.0 mg/ml and solubilized with 1% v/v Triton-X-100 for one hour at room temperature, then centrifuged again at 55,000 rpm for 45 minutes. Pellets were again collected and resuspended in buffer A to 3.0 mg/ml and solubilized overnight at 4oC with 1% lauryldimethyl amine-N-oxide (LDAO). Solubilized lysate was then ultracentrifuged a final time at 55,000 rpm for 45 minutes and the supernatant was collected. Supernatant was passed through a 5 mL column containing Ni2+ sepharose high performance beads equilibrated with a buffer containing buffer A supplemented with 0.1% LDAO. Flowthrough was collected and column was re-equilibrated with buffer. FhuA was eluted using an elution buffer of buffer A supplemented with 0.1% LDAO and 600 mM imidazole. FhuA was then subjected to strong anion exchange on a 1 mL HiTrap Q FF ion exchange column equilibrated with buffer B supplemented with 0.1% LDAO. Bound FhuA was eluted using an elution buffer of buffer A supplemented with 0.1% LDAO and 1M NaCl using an elution gradient of 5 column volumes.     2.5 Colicin M expression  Colicin M was expressed from the E. coli mutant strain BW25113 ?FhuA::CmR from the plasmid pMLD189, which expresses a colicin M with an N-terminal hexhistidine-tag (both the plasmid and strain were donated by Dr. Dominique Mengin-Lecreulx) (Ghachi et al 2006). Cells 29were grown in LB broth supplemented with 80 ?g/ml ampicillin to OD600  ~0.6 and then induced with 1mM IPTG. Cells were collected by centrifugation and and resuspended in buffer A prior to supplementation with 1mM PMSF and then lysed by three passages through a microfluidizer. Cleared lysate was passed through a Ni2+ NTA chromatography column equilibrated with loading buffer A supplemented with 0.1% ?-mercaptoethanol. Colicin M was then eluted with a 10 column volume elution gradient of buffer A with 600mM imidazole and 0.1% ?-mercaptoethanol. 2.6 Nanodisc FhuA reconstitution.  Membrane scaffold protein (MSP) L156 was already available in the lab. Purified L156 was combined with TSGD buffer of 50 mM Tris-HCl, pH = 7.9, 100 mM sodium chloride, 2% glycerol, and 0.1% dodecyl-?-D-maltopyranoside (DDM). Purified FhuA protein was then incubated with MSP L156 at a molecular ratio of 1 nmol of FhuA to 5 nmol of MSP L156 in a 300 ?l volume. Reconstitution was initiated by addition of 80 ?l of biobeads (Bio-Rad) followed by gentle rocking for 16 h at 4?C to slowly adsorb detergent. Beads were removed by low speed centrifugation and collection of the supernatant.  To remove aggregates and free unincorporated MSP the mixture was subjected to gel filtration on a Superdex 200 HR 10/30 column (Amersham) equilibrated with buffer A. Fractions were analyzed by either 4-12% blue- or colourless-native gels or 12% SDS-PAGE and peak fractions were pooled, concentrated using a 30 kDa MWCO centrifugal filter (Millipore, USA), and stored at -80?C.302.7 Isothermal titration calorimetry.  Isothermal titration calorimetry (ITC) is a biophysical technique that provides a variety of thermodynamic parameters. ITC can yield the binding affinity, dissociation constant, stoichiometry, Gibbs free energy, enthalpy, and entropy of an interaction. ITC has been typically used for protein and small molecule interactions but is also used for protein-protein interactions. An ITC instrument consists of two separate cells, an experimental cell and a control cell. The experimental cell contains a protein or protein complex while the control cell contains only water or buffer. A ligand or other protein is then titrated into the experimental cell in precise volumes and the heat of the interaction is measured against the control cell. The heat of the interaction can be exothermic, in which heat is released, or endothermic, in which heat is absorbed. The binding can then be graphed and, using the Origin 7.0 software, the various thermodynamic parameters can be ascertained.  ITC experiments were conducted in a MicroCal ITC-200 apparatus (GE Healthcare) thermostated at 25?C. Samples were dialyzed against 50 mM Tris-HCl (pH=7.9), 100 mM NaCl (plus 2mM ?-mercaptoethanol for experiments involving colicin M) for at least 16 hours, using a 12,000-14,000 Da molecular mass cutoff  dialysis membrane (SpectraPor). Following dialysis, proteins were concentrated using an Amicon Ultra centrifugal concentrator (Millipore) with a 10 kDa molecular cutoff. The quality of the material was tested by colorless native PAGE analysis prior to performing the titration. For the experiments including siderophore, Nd-FhuA was incubated with a 10-fold molar excess of ferricrocin for one hour at 4?C prior to dialysis. The complex of Nd-FhuA with ColM was isolated by gel filtration on a Superdex 200 HR 10/30 column and dialyzed against buffer A plus 2mM ?-mercaptoethanol.  For the experiments 31involving TonB, titrations consisted of 40 time 1?l injections of 95 ?M TonB into a cell containing 12 ?M Nd-FhuA or Nd-FhuA-Fc. In the experiment to determine TonB binding to Nd-FhuA-ColM 200 ?M of TonB was injected into a cell containing 16 ?M of Nd-FhuA-ColM complex. For the experiments involving ColM, titrations consisted of 20 injections of 500 ?M colicin M into a cell containing 25 ?M Nd-FhuA. 500 ?M colicin M was injected into a cell containing 25 ?M Nd-FhuA-Fc to evaluate ferricrocin inhibition of colicin M binding. The heat of dilution was obtained either by injection of ligand into a cell containing only buffer or from injection of ligand into cell after binding site saturation. Stoichiometry of interaction (N), association constant (Keq; KD = 1/Keq), and enthalpy changes (?H) were analyzed using a single-site binding model and errors were derived from chi-squared degrees of freedom on Origin 7 software (MicroCal, Inc.) (Freyer and Lewis, 2008). The Keq value can be determined from the Equation 2-1: Equation 2-1: Equilibrium association constant Wherein Protein A and Protein B form a complex. The stronger the complex between Protein A and Protein B, the larger the Keq value. The Keq constant also reflects the two rate values, Kon and Koff. The dissociation constant, KD, reflects the reciprocal of the Keq value (KD = 1/Keq). Both Keq and KD are constants that represent the equilibrium of association and dissociation, respectively. Both Keq and KD  reflect the specific rates of association (Kon) and dissociation (Koff) such that Keq = Kon/Koff and KD = Koff/Kon. The Kon is measured per moles per second (M-1s-1) while Koff is measured in per second (s-1). These rates show the assembly and 32disassembly of protein complexes over time. Two protein complexes may, therefore, have a similar Keq and KD but may have very different Kon and Koff values. Both the association and dissociation rates are typically very high, indicating that complex formation between macromolecules occurs within microseconds of co-incubation. 2.8 Analytical gel filtration Proteins were applied to a Superdex 200 HR 10/30 column equilibrated with buffer A. The flow rate for experiments was kept at 0.500 ml/min. For experiments with iron-siderophore the Nd-FhuA was incubated with a 10-fold molar excess of ferricrocin for one hour at 4?C to ensure formation of a ligand-loaded Nd-FhuA prior to injection. 2.9 Multiangle light scattering Samples were applied to a Superdex 200 HR 10/30 column equilibrated with buffer A and connected in-line to a MiniDAWN TREOS multi-angle light scattering apparatus and an Optilab T-rEX differential refractive index apparatus (Wyatt Technologies). Samples for light scattering were run at 0.400 ml/min. All data was collected in real-time using the ASTRA V software (Wyatt Technologies) and the molecular mass was calculated using a Debye fit method.  2.10 Trypsinization of Nanodisc FhuA 6?g of purified nanodisc reconstituted FhuA (1-3mg/ml) was incubated in the presence or absence of 0.3?g of ferricrocin, 4?g of colicin M, or 5ug of TonB at 37?C for varying times (5 minutes to 4 hours) with tosylamide phenylethyl chloromethyl ketone-treated trypsin. The 33reaction was halted by addition of SDS electrophoresis buffer and boiling at 95?C for 5 minutes. Samples were then visualized on 10% SDS-PAGE gels by Coomassie brilliant blue stain. Samples were also visualized by transferring bands to nitrocellulose membrane and blotting with a mouse anti-His antibody. 2.11 Other methodsProtein concentrations were determined using the Bradford assay (Bradford, 1976).  Blue-native and clear-native gels, as well as electrophoresis conditions, were as described in Dalal and Duong (2010). All native gels used in this study were 4-12% gradient gels while all SDS-PAGE gels were 12% unless otherwise indicated.  Molecular weight markers used in native PAGE electrophoresis were BSA (67/134 kDa) and ferritin (440 kDa).343. Results:3.1 Incorporation of FhuA into Nanodiscs The FhuA protein was reconstituted into nanodiscs using a membrane scaffold protein (MSP) of 156 amino-acid residue lengths that was produced from a deletion of MSP1D1. This construct is referred to as MSP L156. Purified FhuA was incubated with an optimized quantity of MSP L156. After removal of the detergent the nanodisc reconstituted FhuA was analyzed by size exclusion and native gel electrophoresis. Reconstituted nanodisc FhuA was analyzed and fractionated on size exclusion chromatography (Figure 3-1A). Two peaks were observed eluting from size exclusion chromatography and analyzed on 15% SDS-PAGE to find that the largest peak corresponding to an association of FhuA with MSP whereas the later peak corresponded to MSP alone. Nanodisc reconstituted FhuA, hereafter referred to as Nd-FhuA, was determined to be homogeneous and was compared to both detergent solubilized FhuA and MSP L156 (Figure 3-1B). On colorless native gel electrophoresis it was observed that the Nd-FhuA complex migrates into the gel as a distinct homogeneous band while the detergent solubilized FhuA aggregates near the top of the gel. This Nd-FhuA band is also distinguishable from the MSP L156 band, demonstrating that this complex is not composed entirely of membrane scaffold protein (Figure 3-1C). A molecular weight marker of ferritin (440kDa) and bovine serum albumin (BSA) (67/134 kDa) was used to compare the molecular masses of Nd-FhuA and MSP L156 (Figure 3-1C). Blue native PAGE is a native PAGE technique wherein protein and protein complexes are coated in Serva Blue G dye that confers a net negative charge that both allows 35migration of high pI proteins and complexes into the gel and prevents aggregation of membrane proteins. Bands on blue native PAGE gels also shows a higher molecular mass of Nd-FhuA compared to detergent solubilized FhuA. This indicates that the addition of membrane scaffold protein has increased the mass of the FhuA monomer. Additionally, while Nd-FhuA appears monomeric as a single band, detergent solubilized FhuA appears to form oligomers on blue native PAGE. Both the Nd-FhuA and detergent solubilized FhuA bands are distinguishable from the band corresponding to MSP L156 on blue native PAGE (Figure 3-1C).  Taken together these results establish that the FhuA protein is associated with the MSP and that it has been reconstituted into a water soluble particle characteristic of nanodiscs. 36Figure 3-1. Reconstitution of FhuA into Nanodiscs.(A) Nanodisc-reconstituted FhuA (800 ?g) was injected onto a Superdex 200 HR 10/30 column equilibrated in buffer A (50 mM Tris-HCl, pH 7.9, 100 mM NaCl, 2% glycerol). (B) Fractions 11 to 17 were analyzed by 15% SDS-PAGE. For reference, purified FhuA and MSP-L156 are loaded on the same gel. (C) Clear-native and blue-native PAGE analysis of FhuA in nanodisc (lane 1), FhuA in LDAO (lane 2), and MSP-L156 (lane 3). The molecular weight markers are BSA (67/134 kDa) and ferritin (440 kDa).3.2 Siderophore enhances recruitment of TonB to Nanodisc FhuA Previous research has determined that the recruitment of TonB to FhuA is enhanced by the ligand loaded state of FhuA (Moeck et al. 1997; Khursigara et al. 2004). A colourless native PAGE analysis was performed to assess the recruitment of a soluble truncated TonB consisting of residues 33-239 (hereafter referred to as TonB) to Nd-FhuA and to determine the effect of the iron siderophore ligand ferricrocin. Nd-FhuA and TonB control lanes showed the migration of the Nd-FhuA into the gel, whereas TonB did not migrate on clear native-PAGE (Figure 3-2 A, lane 2). TonB was calculated to have an isoelectric point of  approximately 9.6 based on the amino-acid sequence, which would confer a net positive charge to the protein and inhibit migration on the native PAGE gel system (pH=8.8). Nevertheless, incubation of TonB with Nd-FhuA caused a shift in the migration of the Nd-FhuA band and a corresponding decrease in intensity of the band corresponding to Nd-FhuA alone (Figure 3-2 A, lane 3), indicating stronger formation of a complex between Nd-FhuA and TonB.  The effect of ferricrocin ligand on the recruitment of TonB to Nd-FhuA was tested by preincubation of the Nd-FhuA with a five-fold molar excess of ferricrocin. When visualized on native PAGE the shift in the Nd-FhuA band corresponding to formation of a complex with TonB37was still observed; however, this band was more intense than the band observed in the absence of ferricrocin. Additionally, the decrease in intensity in the band corresponding to uncomplexed Nd-FhuA was more apparent when incubated with ferricrocin (Figure 3-2 A, lane 4), which indicates a more stable complex formation between Nd-FhuA and TonB.  To further assess the formation of a complex between Nd-FhuA and TonB a size exclusion analysis was performed. Controls of Nd-FhuA and TonB were individually passed through a a Tricorn 10/300 column packed with Superdex 200 and elution fractions were analyzed by SDS-PAGE. Nd-FhuA and TonB eluted at separate volumes of ~12 mL and ~14 mL, respectively. Incubation of Nd-FhuA with TonB in the absence of ferricrocin showed that the Nd-FhuA and TonB did not co-elute (Figure 3-2, B). This suggests that TonB is not recruited to form a complex with Nd-FhuA when FhuA is not ligand loaded.  After pre-incubation of Nd-FhuA with a molar excess of ferricrocin, TonB was incubated with the ligand-loaded Nd-FhuA and assessed by size exclusion. In this instance both Nd-FhuA and TonB were observed to co-elute as observed on SDS-PAGE (Figure 3-2, B). From this result it can be inferred that the ligand-loaded state of FhuA is a critical parameter in the recruitment of TonB.  Our size exclusion result is in agreement with similar experiments conducted by Moeck and Lettelier, 2001, that isolated a complex of detergent solubilized FhuA with TonB from size exclusion only after FhuA had been ligand loaded. These results indicate that the ligand-loaded state of Nd-FhuA is important for the recruitment of TonB to Nd-FhuA. 38Figure 3-2. Formation of a complex of Nd-FhuA-TonB32-239.(A) Nd-FhuA (5?g) was incubated with TonB (2?g) in absence or presence of ferricrocin. Proteins were analysed by clear-native PAGE followed by Coomassie blue staining. (B) Nd-FhuA (300?g) was mixed with TonB (65?g), in absence or presence of ferricrocin, before separation on a Superdex 200 HR 10/30 column. The gel filtration fractions were analyzed by SDS-PAGE followed by Coomassie blue staining.3.3 Thermodynamics of Nanodisc FhuA and TonB complex  Previous work to characterize the interaction of TonB with FhuA has been performed using a variety of techniques, including analytical ultracentrifugation and surface plasmon resonance (Khursigara et al. 2004; Khursigara et al. 2005). These techniques have given the  binding affinity of TonB for FhuA in the presence and absence of ferricrocin and given the molar ratio of the interaction. To better characterize the role of ferricrocin in the interaction between TonB and Nd-FhuA isothermal titration calorimetry (ITC) was performed. This technique has the advantage of providing a variety of thermodynamic parameters of the interaction (Kapp, entropy, enthalpy, Gibbs free energy, and molar ratio) occurring during the binding of TonB to Nd-FhuA. 39 TonB was titrated into Nd-FhuA in the presence of ferricrocin and was observed to bind to ligand-loaded Nd-FhuA with a high affinity KD of ~200 nM. A single binding-site model was found to best fit the binding of TonB to Nd-FhuA and the N value was found to be approximately 0.98, indicating that a monomer of TonB was binding to the nanodisc reconstituted FhuA. This reaction was exothermic and had a free energy of (?G = -9.1 kcal?mol-1),  which is a product of a favorable enthalpy (?H = -9.2 kcal?mol-1) and a modestly unfavorable entropy (T?S = -0.05 kcal?mol-1). The thermodynamics of this interaction show that nearly the entirety of the binding interaction is due to the enthalpy of the interaction (Figure 3-3, A).  When TonB was titrated into Nd-FhuA in the absence of ferricrocin it failed to show any interactions between TonB and Nd-FhuA. This result indicates that TonB was not forming a complex with the apoprotein state of Nd-FhuA. Only after preincubation of Nd-FhuA with ferricrocin were we able to measure an interaction between TonB and Nd-FhuA (Figure 3-3, B). In agreement with our previous gel filtration results, formation of a complex between Nd-FhuA and TonB was dependent on the ligand-bound state of Nd-FhuA   40Figure 3-3. Binding affinity between Nd-FhuA and TonB32-239.The ITC thermograms show the interaction of Nd-FhuA with TonB in the presence (A) or absence of ferricrocin (B). Raw ITC traces and integrated heats of interactions are presented. Each titration was performed by injecting 1?L of TonB (95?M) into a cell contained Nd-FhuA (12 ?M) or Nd-FhuA-Fc (12?M), 39 times with 2 minutes between injections. Each titration was initiated with a single 0.5?l injection. The thermodynamic parameters are reported in Table 4.3.4 Nanodisc FhuA binds TonB in a 1:1 molar ratio The stoichiometry between FhuA and TonB has previously been investigated after the discovery that TonB is able to form dimers (Chang et al. 2001). Our experiments on the binding of TonB to Nd-FhuA on ITC showed a 1:1 stoichiometry. To ascertain the stoichiometry of interaction between TonB and the nanodisc reconstituted FhuA we employed both cysteine crosslinked dimers of TonB and multi-angle light scattering. 41 A cysteine substitution was introduced into TonB within the linker region between the hexahistidine sequence and the thrombin cleavage site in a similar manner as employed by Khursigara et al. 2004. Upon purification of TonB-cys it was noted that approximately 50% of the protein population was in a dimerized configuration. Both the dimer and monomer of this TonB cysteine mutant were isolated by gel filtration chromatography (Figure 3-4, A). Using colourless native PAGE it can be observed that both the wild-type TonB and the TonB-cys dimer form a complex when incubated with Nd-FhuA. However, the observed complex of Nd-FhuA and TonB shifted to a considerably higher molecular weight when Nd-FhuA was incubated with the crosslinked dimer of TonB (Figure 3-4, B). Upon incubation of Nd-FhuA and either wild-type TonB or monomeric TonB-cys with the reducing agent DTT there was no apparent shift in migration of the complex suggesting that the reducing agent has no effect on formation of the complex. Addition of reducing agent to the complex of Nd-FhuA and the dimeric TonB-cys resulted in a shift in the band migration to a position of lower molecular weight equivalent to the complex of Nd-FhuA and monomeric TonB (Figure 3-4, B). This result shows that incubation of dimerized complex results in a complex of two TonB on one Nd-FhuA; however, incubation together with the reducing agent DTT disrupts the cysteine crosslink and only one monomer of TonB remains bound to Nd-FhuA.      Multi-angle light scattering was performed to further examine the stoichiometry of Nd-FhuA and TonB interactions. This technique offers the benefit of determining the molecular mass of individual proteins as well as protein complexes allowing the stoichiometry to be ascertained. Nd-FhuA and TonB were individually analyzed by multi-angle light scattering and each was determined to be monodisperse. Nd-FhuA was found to have a molecular mass of of ~130 kDa ? 420.4% and TonB was found to have a molecular mass of ~25 kDa ? 5%. The molecular mass of the TonB was calculated to be ~25 kDa based on amino-acid sequence alone, therefore TonB exists in solution as a monomer. Co-elution of ligand-loaded Nd-FhuA and TonB yielded a complex with an apparent molecular mass of ~160 kDa ? 3% (Figure 3-4, C). TonB is, therefore, monomeric in solution and forms a 1:1 stoichiometric complex with nanodisc reconstituted FhuA.43Figure 3-4. Binding stoichiometry between Nd-FhuA and TonB32-239.(A) TonB WT (6?g) and disulfide-linked TonB dimer (6?g) were incubated for 2 minutes at 37?C in the absence (lane 2) and presence (lane 3) of DTT (1mM). Samples were analyzed by SDS-PAGE and Coomassie blue staining. (B) Nd-FhuA (5?g) was incubated with wild-type TonB or disulfide-linked TonB (4?g each) in buffer A. The indicated samples were incubated with DTT (1mM) for 2 minutes at 37?C prior to analysis by clear-native PAGE.(C) Multi-angle light scattering (MALS) analysis of Nd-FhuA, TonB and Nd-FhuA-TonB in the presence of ferricrocin. Proteins (400?g) were loaded on a Superdex 200 HR 10/30 column equilibrated in buffer A. The measured molecular masses are the following: Nd-FhuA ~130kDa (? 0.4%); TonB ~25kDa (? 5%); Nd-FhuA-Fc-TonB ~160kDa (? 3%).443.5 Colicin M binding to Nanodisc FhuA is inhibited by ligand FhuA acts as the cell surface receptor for the antimicrobial protein colicin M. Colicin M has been shown to bind to the extracellular loops of FhuA and may access the periplasm by transversing through the channel of FhuA (Killmann et al. 1995; reviewed by Braun et al. 2012). Colicin M binding to Nd-FhuA was first investigated using native gel electrophoresis.  Complex formation was only observed between colicin M and Nd-FhuA in the absence of ferricrocin (Figure 3-5 A, lane 4). When ferricrocin was preincubated with Nd-FhuA no shift in the band corresponding to Nd-FhuA was observed, indicating formation of complex was inhibited (Figure 3-5A, lane 5). This implies that binding of ferricrocin either blocks the colicin M binding site or non-competitively conformationally alters the colicin M binding site. 3.6 Thermodynamics of Colicin M binding to Nanodisc FhuA  To further characterize the interaction of colicin M with nanodisc FhuA and to observe the effect of ferricrocin the thermodynamics of colicin M binding to Nd-FhuA was examined by isothermal titration calorimetry. In the absence of ferricrocin, the affinity of colicin M to Nd-FhuA was found to be strong with a KD of approximately 3.5 nM. This interaction was found to be an endothermic interaction, with the free energy of the interaction (?G = -11.5 kcal?mol-1) being the result of an unfavorable enthalpy of (?H = 7.9 kcal?mol-1) and a favorable entropy (T?S = 19.5 kcal?mol-1). Because this interaction between colicin M and FhuA is entirely driven by entropy strongly implies a primarily hydrophobic interaction between these proteins (Figure 3-5, B). This is supported by work done by Helbig and Braun in 2011 that shows that the deletion of a hydrophobic helix, ?1, from the receptor binding domain of colicin M reduces binding of 45colicin M to FhuA. FhuA has a number of aromatic residues lining the opening to the antechamber of the protein that are thought to provide the initial attraction for siderophores (Cao and Klebba, 2002, and references therein). It is possible that colicin M binds to FhuA by interactions between hydrophobic regions of the receptor binding domain of colicin M and these aromatic residues of FhuA.        Preincubation of Nd-FhuA with ferricrocin prior to performing ITC with colicin M failed to show any interaction between colicin M and the ligand-loaded Nd-FhuA (Figure 3-5, C). This is in agreement with our previous results on native PAGE and size exclusion chromatography. This inhibition of complex formation between colicin M and Nd-FhuA by ferricrocin reinforces the finding that the binding of siderophore by FhuA conformationally alters FhuA such that colicin M is unable to bind. Previous work has found that the siderophores act to protect cells against a variety of colicins including colicin M (Wayne et al. 1976). Additional work has found that the prospective binding site of colicin M to FhuA is the external gating loop (Killmann et al. 1995). Binding of siderophore to FhuA has been shown to have an effect on the conformation of this gating loop (B?s et al. 1998). Taken together these results suggest a structural basis in the inhibition of binding of colicin M to FhuA by siderophore wherein binding of siderophore to FhuA causes a conformational change in the gating loop preventing binding by colicin M.46Figure 3-5: Ferricrocin prevents binding of Colicin M to Nd-FhuA.(A) Nd-FhuA (5?g) was incubated with ColM (5?g) with or without ferricrocin. The resulting complexes were separated on clear-native PAGE and visualized by Coomassie blue staining. (B-C) ColM (500 ?M) was injected (20 injections; 1?l each) into a cell containing apo-FhuA or ligand-loaded FhuA (25 ?M). Raw ITC traces and integrated heat of interactions are presented. The thermodynamic parameters are reported in Table 4.3.7 Colicin M does not promote binding of TonB to FhuA It has been proposed previously that some colicins utilize molecular mimicry to interact with their receptor. Colicin E3 and Ia have a binding region that mimics the chemistry of the ligands cyanocobalamin and catecholate iron, respectively (Cao and Klebba. 2003, and references therein). The binding of colicin E3 to BtuB causes a change in the conformation of BtuB that alters the TonB box of BtuB (Cadieux et al. 2003; Fanucci et al. 2003a). Colicin M was demonstrated to interact with TonB through the TonB box at the N-terminal end of colicin M. Mutations in the TonB box of colicin M that inhibited transport across the outer membrane were suppressed by mutations in TonB, suggesting a direct interaction between colicin M and TonB (Pilsl et al. 1993). Mutations in the TonB box of FhuA that inhibited recruitment of TonB were also found to inhibit transport of colicin M (Sch?ffler and Braun, 1989). Taken together 47these past observations suggest that colicin M may behave as a molecular mimic of hydroxyamate iron chelators and may cause recruitment of TonB to FhuA.  The formation of a ternary complex between FhuA, TonB, and colicin M has not been thoroughly investigated in vitro, investigation of this complex using nanodisc reconstituted FhuA was performed using native gel electrophoresis, size exclusion chromatography, and isothermal titration calorimetry.  Colourless native PAGE analysis of colicin M incubated with Nd-FhuA and TonB revealed a ternary complex on native gel (Figure 3-6, A lane 4). This complex was found to be disrupted by the addition of ferricrocin to the complex, in agreement with previous results on native PAGE, and resulting in formation of a complex between Nd-FhuA and TonB while colicin M was excluded entirely (Figure 3-6, A lane 5).  Size exclusion chromatography analysis was performed on mixture of Nd-FhuA, colicin M, and TonB. It was found that, while Nd-FhuA and colicin M eluted together as a complex from the size exclusion column, TonB eluted separately from the Nd-FhuA-colicin M complex (Figure 3-6, B). This  indicates that no stable ternary complex was formed and that colicin M, unlike ferricrocin, is unable to promote formation of a complex between Nd-FhuA and TonB.  Isothermal titration calorimetry of TonB injected into a isolated complex of Nd-FhuA and colicin M also showed no reaction between TonB and the Nd-FhuA-colicin M complex (Figure 3-6, C). This indicates that colicin M, unlike ferricrocin, did not promote the binding of TonB to Nd-FhuA. This result indicates that neither TonB box, either on FhuA or on colicin M, was accessible or in a conformation capable of recruiting TonB. 48 These results demonstrate that colicin M binding to FhuA does not induce conformational changes in FhuA that would facilitate recruitment of TonB to FhuA. Figure 3-6: Colicin M does not seem to trigger the binding of TonB to Nd-FhuA.(A) Nd-FhuA (6?g), TonB (4?g), and colicin M (4?g) were incubated together in buffer A. The mixture was analyzed by clear-native PAGE. Where indicated, Nd-FhuA was pre-incubated with ferricrocin. (B) The mixture in (A) was applied on a Superdex 200 HR 10/30 column equilibrated in buffer A. The gel filtration fractions were analyzed by SDS-PAGE.(C) TonB (200 ?M) was injected into a cell containing the complex Nd-FhuA-ColM (16 ?M). The raw ITC traces and integrated heats of the interaction are presented. The thermodynamic parameters are reported in Table 4.49Table 3.1: Isothermal titration calorimetry resultsTitrant Cell N Kd (nM) ?H (cal/mol)?S (cal/mol/deg)TonB Nd-FhuA apo - - - -TonB Nd-FhuA-Fc 0.977?0.0100 200.4?29.3 -9186?133.4 -0.164Colicin M Nd-FhuA apo 0.906?0.0016 3.48?1.09 7969?33.3 65.4Colicin M Nd-FhuA-Fc - - - -TonB Nd-FhuA-ColM - - - -The estimated errors are based on a ?2 minimized fit of the experimental data to a single-site binding model using Origin 7.0 software (OriginLab).3.8 Conformational changes in FhuA caused by ligand and Colicin M It has previously been demonstrated that FhuA undergoes conformational changes upon becoming ligand loaded (Moeck et al. 1996). Utilizing a modified trypsinization technique developed by Moeck et al. I was able to investigate the conformational changes of FhuA reconstituted in nanodisc in complex with ferricrocin or colicin M.  Samples of Nd-FhuA were incubated at 37?C in the presence or absence of ferricrocin with trypsin for varying times. Samples were then boiled with SDS sample buffer and visualized on SDS-PAGE. Results show that FhuA is progressively degraded into multiple bands in comparison to trypsin-free controls in the absence of ferricrocin indicating that multiple sites of trypsin cleavage are accessible in the apoprotein state of FhuA. However, in the presence of ferricrocin the digestion pattern of FhuA changes to no longer yield the multiple digestion products found in the apoprotein digestions (Figure 3-7, A). This shows that the trypsin 50accessible cleavage sites on FhuA become inaccessible upon the formation of ligand-loaded complex.  This trypsinization technique was then applied to probe the possible conformational changes induced in FhuA by interactions with colicin M and TonB. Trypsinization of Nd-FhuA with TonB produced the same resultant multiple cleavage products as Nd-FhuA alone indicating that either TonB is not binding to Nd-FhuA or that binding of TonB to FhuA does not induce a conformational change in FhuA that impact trypsin cleavage sites (Figure 3-7, B lane 5). A control of Nd-FhuA incubated with TonB and ferricrocin produced that same digestion pattern as ferricrocin alone, however this result is most likely to be the result of ferricrocin induced conformational changes in Nd-FhuA rather than any effect of TonB (Figure 3-7, B lane 6). Trypsinization of Nd-FhuA in the presence of colicin M produced a similar digestion pattern as with ferricrocin, indicating that binding of colicin M to FhuA induces a similar conformational change in FhuA as ferricrocin possibly to mimic a ligand-loaded state (Figure 3-7, B lane 4). It is also a possibility that colicin M is bound to the extracellular surface loops of FhuA such that certain trypsin cleavage sites are protected from digestion by trypsin. To test this possibility a western blot was performed using a anti-hexahistidine antibody. All degradation products of FhuA were visible on the western blot as on SDS-PAGE (Figure 3-7, C). The hexahistidine tag on FhuA is located at position 405 of the protein near to the gating loop. If trypsinization of FhuA results in cleavage of extracellular loops or compromises the ?-barrel of FhuA it might be expected that some of the larger degradation products visualized on SDS-PAGE will not appear on western blot. If cleavage primarily occurs near the N-terminus or in the plug domain then most of the cleavage products will be visualized by western blot. Because all degradation 51products appeared on western blot it can be interpreted that trypsin cleaves at the N-terminus and not within the ?-barrel. To further test the possibility that colicin M is concealing trypsin cleavage sites rather than inducing conformational changes, a titration of trypsin digestion was performed in which a molar excess of trypsin was added to complexes of Nd-FhuA and colicin M (Figure 3-7, D). If colicin M was concealing trypsin accessible sequences on FhuA a molar excess of trypsin would digest colicin M and expose to the accessible sites. Nevertheless, results show that despite the excess of trypsin FhuA is still not digested into the same pattern as a ligand unloaded Nd-FhuA.  There remains the possibility that trypsin was inhibited by the presence of ferricrocin or colicin M. To test this possibility control experiments using the maltose ABC transporter FGK reconstituted into nanodiscs showed that trypsinization of NdFGK was not inhibited by either ferricrocin or colicin M and that digestion of Nd-FhuA occurs even in the presence of Nd-FGK and is still inhibited by the addition of ferricrocin or colicin M (Figure 3-7, E). 5253Figure 3-7: Colicin M and Ferricrocin affect the conformation of Nanodisc reconstituted FhuA. (A) Nd-FhuA was incubated with trypsin at 37?C for the indicated times in the presence or absence of ferricrocin. Samples were resolved on 10% SDS-PAGE and visualized by Coomassie blue staining. (B) Nd-FhuA was incubated with trypsin at 37?C for 30 minutes in the presence or absence of indicated binding partner ferricrocin ligand, colicin M, and TonB. (C) Western blot analysis of Nd-FhuA digestion using anti-His tag antibody. (D) A fixed amount of Nd-FhuA (5 ?g) and colicin M (2 ?g) was incubated with an increasing amount of trypsin at 37?C for 1 hour. (E) Nd-FGK was incubated with trypsin at 37?C for 30 minutes in the absence or presence of ferricrocin, Nd-FhuA, and colicin M.544. DiscussionDifferent types of detergent have been shown to affect the conformation of the TonB-dependent transporter BtuB (Fanucci et al. 2003b). To negate these potential problems the TonB-dependent transporter FhuA was reconstituted into nanodiscs. Nanodiscs allow investigation of membrane proteins without the requirement of detergents or liposomes (Bayburt et al. 2002; Bayburt and Sligar, 2003). In this study, a combination of native gel electrophoresis, analytical gel filtration, multi-angle light scattering, and isothermal titration calorimetry were used to characterize interactions of the nanodisc reconstituted TonB-dependent transporter FhuA with TonB and colicin M. 4.1 Reconstitution of FhuA into Nanodiscs The incorporation of FhuA into nanodiscs offers the prospect of conducting binding studies of FhuA with its cognate substrates and binding partners. Previous work has been conducted using membrane mimetic environments such as detergents (Khursigara et al 2004; 2005) and lipid vesicles (Kim et al. 2007). As demonstrated previously detergents can affect the TonB box of TonB-dependent transporters (Fanucci, 2003b) and the type of detergent can alterinteractions with TonB (Choul-Li et al. 2008). Reconstitution of proteins into vesicles results in large micron-sized vesicles and heterogeneity, which complicate binding studies. Reconstitution of FhuA into monodisperse nanodiscs negates these inherent problems of these methods.  Reconstitution of FhuA into nanodiscs also demonstrates the possibility of other TonB dependent transporters being reconstituted into nanodiscs for evaluation in binding studies with TonB and their respective colicins. 554.2 Recruitment of TonB to Nanodisc FhuA FhuA interacts with TonB to transport bound ligands into the periplasm. Previous research has found using surface plasmon resonance (SPR) that the ligand-loaded state of FhuA enhances the recruitment of TonB (Khursigara et al. 2004). On colourless native PAGE it was observed that the nanodisc reconstituted FhuA interacts with TonB and that the incubation of reconstituted FhuA with ferricrocin prior to incubation with TonB resulted in a more stable complex in agreement with these previous results. Because recruitment of TonB to FhuA was retained in nanodiscs we assume that the structural conformation of FhuA are retained in nanodiscs. Further characterization of the TonB interaction with nanodisc FhuA by analytical gel filtration showed similar results to Moeck and Lettelier, 2001. TonB did not form a complex with nanodisc reconstituted FhuA unless ferricrocin was present. This observation indicates that TonB will not form a complex with FhuA unless FhuA is ligand-loaded. Previous studies conducted on detergent solubilized FhuA report that TonB forms a complex with FhuA in the absence of ligand (Khursigara et al. 2004; Khursigara et al. 2005). It is possible that detergents in these studies have facilitated the interaction between FhuA and TonB. In nanodiscs, FhuA is retained in a native conformation and interactions with TonB cannot be facilitated by detergents.   Isothermal titration calorimetry was performed to further characterize the interaction between nanodisc reconstituted FhuA and TonB. The binding of TonB to ligand-loaded nanodisc FhuA was a primarily enthalpy driven interaction, TonB interacts with FhuA at the at the constitutively unfolded TonB box (Locher et al. 1998; Ferguson et al. 1998; Kim et al. 2007). 56Strand exchange occurs between the C-terminus of TonB and the TonB box of FhuA (Pawelek et al. 2006). Strand exchange interactions involve unfolded protein disorder-to-order interaction and, therefore, are driven primarily by enthalpy contributions from the interacting partners (Dyson and Wright, 2005). In agreement with results obtained by analytical gel filtration, the apo-protein of FhuA failed to show an interaction with TonB, again indicating that FhuA in the nanodisc may not interact with TonB when FhuA is not ligand loaded. The dissociation constant between FhuA and TonB as measured by ITC when ligand-loaded was approximately 200nM, a moderate difference between the constant as determined by other studies (Khursigara et al 2004; Freed et al. 2013). 4.3 The stoichiometry between Nanodisc FhuA and TonB The stoichiometry of TonB for TonB-dependent transporters has been investigated since it was determined that certain truncated TonB constructs formed dimers (Chang et al. 2001). Early work in this area using analytical ultracentrifugation has shown that TonB also forms dimers when in complex with FhuA (Khursigara et al. 2004; Khursigara et al. 2005). More recent research using site-directed spin labeling has proposed that a significant population of TonB forms dimers but convert to monomer when a complex is formed with TonB-dependent transporters, such as FhuA or BtuB (Freed et al. 2013). Interpretation of our ITC data yielded a monomeric interaction of TonB with nanodisc FhuA according to the single-site binding model leading us to further investigate the stoichiometry of TonB and nanodisc FhuA interactions.  Interactions of nanodisc reconstituted FhuA with wild-type and dimeric cysteine-mutant TonB reveal a difference in complex size when visualized on colourless native gel. The 57disruption of the dimeric TonB with the reducing agent DTT caused the formation of a Nd-FhuA-TonB complex identical to wild-type TonB (Figure 3, A). This indicates that the wild-type TonB binds as a monomer to the nanodisc reconstituted FhuA.    To further investigate the interaction a multi-angle light scattering analysis was performed to determine the molecular mass of both the individual nanodisc and TonB as well as the complex (Figure 3, B). This analysis showed that the complex of TonB with nanodisc reconstituted FhuA contained a monomer of TonB, in agreement with the native PAGE analysis.  These results, taken together, indicate that TonB interacts with FhuA as a monomer and that dimerization of TonB does not occur during the interaction with ligand-loaded TonB-dependent transporters. 4.4 Colicin M interactions with Nanodisc FhuA A crystal structure of colicin M in complex with FhuA is not currently available. The endothermic binding of colicin M to nanodisc FhuA, with a very favorable positive entropy and a unfavorable enthalpy, points to a hydrophobic interaction. This result suggests the association of hydrophobic surfaces disrupts highly structured water cages surrounding the hydrophobic regions and causes a corresponding increase in entropy. It has been proposed that these hydrophobic interactions between proteins allow further interactions such as hydrogen bonding and electrostatic interactions to occur (Ross and Subramanian, 1981). Prior work has proposed that the highly hydrophobic helix ?1 of colicin M (residues 38-46) functions to adhere colicin M to the cytoplasmic membrane to access substrate (Zeth, et al. 2008). It was shown that the deletion of the ?1 helix results in both significantly reduced uptake of colicin M into cells and an 58inability to compete against albomycin (Helbig and Braun, 2011). Therefore it appears that helix ?1 acts to produce a hydrophobic interaction between colicin M and FhuA to facilitate binding of colicin M to FhuA. The largely aromatic nature of the opening of the FhuA antechamber revealed from the crystal structure reinforces the importance of hydrophobic interactions both for the initial recruitment of iron siderophores as well as for the binding of colicin M.  Binding of colicin M to nanodisc FhuA was a strong interaction at less than 10nM. Under optimal conditions it has been found that some other colicins have equivalently strong binding to their respective receptors such as colicin E9 (Housden et al. 2005) and colicin E3 (Kurisu et al. 2003) for the receptor BtuB and colicin Ia for the receptor Cir (Konisky and Cowell, 1972). The most parsimonious explanation for the strong binding of colicin M for FhuA is the relatively low number of FhuA copies per cell (~1000 per cell) limits the availability of both the receptor and transporter of colicin M (Schultz et al. 1989). High affinity binding is therefore required to ensure that colicin M adsorbs to the surface of target cells despite the rarity of the necessary transporter. Because binding of the ligand ferricrocin to FhuA acted to inhibit the binding of colicin M to FhuA suggests that the residues responsible for binding of colicin M are sequestered by ferricrocin. The lack of a crystal structure of colicin M in complex with FhuA hampers efforts to understand the contributions of individual residues as well as conformational changes occurring in both colicin M and FhuA upon formation of a complex. It has been demonstrated by previous work that conformational change causes colicin M to become trypsin sensitive (Schaller et al. 1981). Based on the apparent hydrophobic interaction, previous work on deletion of the ?1 helix, and conformational change in colicin M upon binding of FhuA indicates that colicin M may 59extend the receptor binding domain helix ?1 into the opening of the FhuA antechamber where the hydrophobic residues of the ?1 helix can interact with the hydrophobic aromatic residues within the barrel. When FhuA is ligand loaded with ferricrocin these hydrophobic and aromatic residues either contribute to the binding of the ligand, which would disagree with the crystal structure of FhuA in complex with siderophore ligand, or undergo conformational changes in FhuA, either scenario effectively sequesters the residues that are required for binding of colicin M. Results demonstrating that colicin M also induces conformational changes in FhuA similar to changes elicited by ferricrocin may also indicate the involvement of these hydrophobic residues. 4.5 Ternary complex of FhuA, TonB, and Colicin M Previous studies have investigated the conformational changes induced on receptors by the binding of colicins (Cadieux et al., 2003; Fanucci et al., 2003a; Devanathan and Postle, 2007). Colicin B has been shown to induce conformational changes in the globular plug domain of FepA that require an interaction with TonB (Devanathan and Postle, 2007). The receptor binding domain of colicin E3 also induces conformational changes in the Ton box of BtuB that reduce the affinity of TonB for BtuB (Cadieux et al., 2003; Fanucci et al., 2003a, Freed et al. 2013). Site-directed mutations introduced into FhuA have demonstrated that the TonB box of FhuA is required for transport of colicin M (Sch?ffler and Braun, 1989).  Observations here show that recruitment of TonB to FhuA is not supported by the binding of colicin M. This indicates that the binding of colicin M to FhuA either does not induce a major conformational change in FhuA to recruit TonB or that any conformational changes induced in FhuA by the binding of colicin M are such that the Ton box is made unavailable for the binding 60of TonB. Trypsinization of FhuA in complex with colicin M showed similar conformation alterations to those made by ferricrocin; however, these changes are such that TonB is not recruited to FhuA for transport.  One model of colicin M transport has the binding of colicin M to FhuA acting to recruit TonB to discharge the plug domain out of the lumen of the ?-barrel allowing colicin M to enter the channel and access the periplasm (Zeth et al. 2008). Conformational changes in FhuA caused by the binding of colicin M were investigated previously by labeling of cysteine residues introduced into the plug domain of FhuA in the presence or absence of colicin M; however, these experiments showed that the plug domain is not dislocated from the lumen of the ?-barrel (Braun et al. 2012). The inability to dislocate the plug from the channel of FhuA implies that colicin M may not transit through FhuA. Taken together with our results to show that colicin M does not promote the recruitment of TonB to FhuA it may be possible that colicin M does not transit through FhuA and, instead, commandeers an outer membrane porin to be transported into the periplasm in much the same way that colicin E3 utilizes BtuB as a receptor and OmpF as a transporter. 615. Conclusion Nanodiscs have been employed previously to characterize a variety of membrane proteins in binding studies including the maltose permease MalFGK2 and the protein translocon SecYEG and interactions with their cognate soluble binding partners. However, nanodiscs have not previously been employed to study the interactions of TonB dependent transporters and TonB. We report the reconstitution of FhuA into nanodiscs and we show the dependence of the interactions with TonB on the presence of the ligand ferricrocin. The equilibrium binding constant of Nd-FhuA with TonB when ligand-loaded is ~200nM in an exothermic interaction and the stoichiometry of interaction was found to be 1:1 using both dimeric TonB and multiangle light scattering.  FhuA in nanodiscs also forms a complex with colicin M.  The equilibrium binding constant between Nd-FhuA and colicin M was observed to be ~3.5 nM in an endothermic interaction and ferricrocin renders FhuA refractory to the colicin, in agreement with earlier observations in vivo.  626. Future directions Nanodisc reconstitution is an approach that should be applied to better characterize other TonB dependent transporters, such as BtuB and FepA. Because of the differences in TonB binding affinity between FhuA reported in detergent and FhuA in nanodiscs it is probable that the binding affinities and stoichiometry reported for other TonB dependent transporters in detergents will also be different in nanodiscs. While FhuA signals the ligand-loaded state with unwinding of a switch helix, other TonB-dependent transporters, such as BtuB, do not have a switch helix and signal the ligand-loaded state using a different mechanism (Xu et al. 2006). The different signaling mechanisms of TonB-dependent transporters should be assessed using nanodisc reconstitution. Additionally, the discovery that colicin M binding to FhuA in nanodiscs is not sufficient to induce the recruitment of TonB to FhuA implies that there are additional details of the colicin M transport mechanism that remain uncharacterized. It will be important to determine if additional interacting proteins, such as a second FhuA, are involved in subsequent steps in the transport process. Colicin M could also act as a vehicle to deliver cargo to the periplasm of cells, an approach used by other groups to deliver toxic proteins to pathogenic bacteria (Lukacik et al. 2012). Additionally, it is now possible to study inner and outer membrane protein complex interactions, an outward facing inner membrane vesicle or sphereoplasts (reviewed by Postle, 2007) could be developed with a PMF to chart interactions with nanodisc reconstituted TonB-dependent transporters in the absence of detergents. 63References: Adams, H., Zeder-Lutz, G., Schalk, I., Pattus, F., and H. Celia. 2006. Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa. J. Bacteriol. 188: 5752-5761. Alami, M., Dalal, K., Lelj-Garolla, B., Sligar, S. G., and F. Duong. 2007. Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA. EMBO J. 26: 1995-2004. Bao, H., and F. Duong. 2012. Discovery of an auto-regulation mechanism for the maltose ABC transporter MalFGK2. Plos ONE. 7: e34836. Bathaie, S. Z., Moosavi-Movahedi, A. A., and A. A. Saboury. 1999. Energetic and binding properties of DNA upon interaction with dodecyl trimethylammonium bromide. Nucleic Acids Res. 27:1001?1005. Bayburt, T. H., Grinkova, Y. V., and S. G. Sligar. 2002. Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold protiens. Nano Letters 2: 853-856. Bayburt, T. H., and S. G. Sligar. 2003. Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers. Protein Sci 12: 2476?2481.? Bayburt, T. H., Grinkova, Y. V., and S.G. Sligar. 2006. Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs. Arch. Biochem. Biophys. 450: 215-222. B?s, C., Lorenzen, D., and V. Braun. 1998. Specific In Vivo Labeling of Cell Surface-Exposed Protein Loops: Reactive Cysteines in the Predicted Gating Loop Mark a Ferrichrome Binding Site and a Ligand-Induced Conformational Change of the Escherichia coli FhuA Protein. J. Bacteriology. 180(3): 605-613. ? Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding". Anal. Biochem. 72: 248?54. Braun, V., K. Schaller, and H. Wolf. 1973. A common receptor protein for phage T5 and colicin M in the outer membrane of Escherichia coli B. Biochim. Biophys. Acta 323:87?97. Braun, V., S. Frenz, K. Hantke, and K. Schaller. 1980. Penetration of colicin M  into cells of Escherichia coli. J. Bacteriol. 142: 162-168. Braun, V., Hantke, K., and W. K?ster. 1998. in Metal Ions in Biological Systems (Sigel, A., and Sigel, H., eds) 35: pp. 67?145, Marcel Dekker, Inc., New York.64 Braun, M., F. Endriss, H. Killmann, and V. Braun. 2003. In vivo reconstitution of the FhuA transport protein of Escherichia coli. J. Bacteriol. 185: 5508-5518. Braun, V. 2009. FhuA (TonA), the Career of a Protein. J. Bacteriol. 191: 3431-3436. Braun, V., Helbig, S., and S. I. Patzer. 2012. Import of periplasmic bacteriocins targeting the murein. Biochem. Soc. Trans. 40: 1449-1455. Brillet, K., Journet, L., C?lia, H., Paulus, L., Stahl, A., Pattus, F., and D. Cobessi. 2007. A ?-strand lock exchange for signal transduction in TonB-dependent transducers on the basis of a common structural motif. Structure 15: 1383-1391.? Buchanan, S.K.; Smith, B.S.; Venkatramani, L., Xia, D., Esser, L., Palnitkar, M., Chakraborty, R., van der Helm, D., and J. Deisenhofer. 1999. Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Natural Structural Biology, 6: 56-63. Cadieux, N., Phan, P. G., Cafiso, D. S., and R. J. Kadner. 2003. Differential substrate-induced signaling through the TonB-dependent transporter BtuB. Proc Natl Acad Sci USA 100: 10688-10693.	 Carmel, G., and J. W. Coulton. 1991. Internal deletions in the FhuA receptor of Escherichia coli K-12 define domains of ligand interactions. J. Bacteriol. 173: 4394-4403. Carter, D. M., Miousse, I. R., Gagnon, J.-N., Martinez, E., Clements, A., Lee, J., Hancock, M. A., Gagnon, H., Pawelek, P. D., and J. W. Coulton. 2006. Interactions between TonB from Escherichia coli and the periplasmic protein FhuD. J. Biol. Chem. 281: 35413-35424. Cascales, E., Lloub?s, R., and J. N. Sturgis. 2001. The TolQ-TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA-MotB. Mol. Microbiol. 42: 795?807. Cascales, E., Buchanan, S. K., Duche, D., Kleanthous, C., Lloubes, R., Postle, K., Riley, M., Slatin, S., and D. Cavard. 2007. Colicin biology. Microbiol. Mol. Biol. Rev. 71: 158-229. Cao, Z. and P. E. Klebba. 2002. Mechanisms of colicin binding and transport through outer membrane porins. Biochimie 84: 399-412. Chang, C., Mooser, A., Pl?ckthun, A., and A. Wlodawer. 2001. Crystal structure of the dimeric C-terminal domain of TonB reveals a novel fold. J. Biol. Chem. 276: 27535-27540.65 Chakraborty, R., E. Storey, D., and van der Helm. 2006. Molecular mechanism of ferricsiderophore passage through the outer membrane receptor proteins of Escherichia coli. Biometals 20: 263-274. Chimento, D. P., Mohanty, A . K., Kadner, R. J., and M. C. Wiener. 2003. Substrate-induced transmembrane signaling in the cobalamin transporter BtuB. Nat. Struct. Biol. 10: 394-401. Chimento, D. P., Kadner, R. J., and M. C. Wiener. 2005. Comparative Structural Analysis of TonB-Dependent Outer Membrane Transporters: Implications for the Transport Cycle. Proteins 59: 240-251. Chin, J. W., and P. G. Schultz. 2002. In vivo photocrosslinking with unnatural amino acid mutagenesis. Chembiochem. 3: 1135-1137. Choul-Li, S., Adams, H., Pattus, F., and H. Celia. 2008. Visualization of interactions between siderophore transporters and the energizing protein TonB by native PAGE. Electrophoresis 29: 1333-1338. Cobessi, D., H. Celia, N. Folschweiller, I.J. Schalk, M.A. Abdallah, and F. Pattus. 2005. The crystal structure of the pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 resolution. J. Mol. Biol. 347: 121-134. Dalal, K., and F. Duong. 2010. Reconstitution of the SecY translocon in nanodiscs. Methods Mol. Biol. 619: 145?156. Davidson, A. L., and J. Chen. 2004. ATP-binding cassette transporters in bacteria. Annu. Rev. Biochem. 73:241-268. Denisov, I. G., Grinkova, Y. V., Lazarides, A. A., and S. G. Sligar. 2004. Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J. Am. Chem. Soc. 126: 3477?3487. Devanathan, S., and K. Postle. 2007. Studies on colicin B translocation: FepA is gated by TonB. Mol. Microbiology 65: 441-453. Denisov, I. G., and S. G. Sligar. 2011. Cytochromes P450 in nanodiscs. Biochim. Biophys. Acta. 1814: 223-229. Dreher, R., Braun, V., and B. Wittmann-Liebold. 1985. Functional domains of colicin M. Arch. Microbiol. 140: 343-346.66 Dyson, H. J., and P. E. Wright. 2005. Intrinsically unstructured proteins and their functions. Nature Rev. Mol. Cell Biol. 6: 197-208. Eisenhauer, H. A., Shames, S., Pawelek, P. D., and J. W. Coulton. 2005. Siderophore Transport through Escherichia coli Outer Membrane Receptor FhuA with Disulfide-tethered Cork and Barrel Domains. J. Biol. Chem. 280: 30574-30580. Endri?, F., M. Braun, H. Killmann, and V. Braun. 2003. Mutant analysis of the Escherichia coli FhuA protein reveals sites of FhuA activity. J. Bacteriol. 185: 4683?4692. Fanucci, G. E., Cadieux, N., Kadner, R. J., and D. S. Cafiso. 2003. Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter. Proc Natl Acad Sci USA 100: 11382-11387. Fanucci, G. E., Lee, J. Y., and D. S. Cafiso. 2003. Membrane mimetic environments alter the conformation of the outer membrane protein BtuB. J Am Chem Soc 125: 13932-13933. Faraldo-G?mez, J. D., Smith, G. R., and M. S. Sansom. 2003. Molecular Dynamics Simulations of the Bacterial Outer Membrane Protein FhuA: A Comparative Study of the Ferrichrome-Free and Bound States. Biophys. 85: 1406-1420. Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K., and W. Welte. 1998. Siderophore-mediation iron transport: crystal structure of FhuA with bound lipopolysaccharides. Science 282: 2215-2220.  Ferguson, A. D., Coulton, J. W., Diederichs, K., and W. Welte. 2001. in Handbook of Metalloproteins in (Messerschmidt, A., Huber, R., Poulos T., and Wieghardt, K., eds) pp. 834?849, John Wiley & Sons, Ltd., Chichester. Ferguson, A. D., J. K?dding, G. Walker, C. B?s, J. W. Coulton, K. Diederichs, V. Braun, and W. Welte. 2001. Active transport of an antibiotic rifamycin derivative by the outer-membrane protein FhuA. Structure 9: 707-716. Ferguson, A. D., R. Chakraborty, B. S. Smith, L. Esser, D. van der Helm, and J. Deisenhofer. 2002. Structural basis of gating by the outer membrane transporter FecA. Science. 295: 1658-1659. Ferguson, A. D., and J. Deisenhofer. 2002. TonB-dependent receptors-- structural perspectives. BBA - Biomembranes 1565: 318-332. Ferguson, A. D., Chakraborty, R., Smith, B. S., Esser, L., Van Der Helm, D. and J. Deisenhofer. 2002. Structural basis of gating by the outer membrane transporter FecA. Science 295: 1715-1719.67 Ferguson, A. D., and J. Deisenhofer. 2004. Metal import through microbial membranes. Cell 116: 15?24. Fischer, E., G?nter, K., and V. Braun. 1989. Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB. J. Bacteriol. 171: 5127-5134. Freed, D. M., Lukasik, S. M., Sikora, A., Mokdad, A., and D. S. Cafiso. 2013. Monomeric TonB and the TonB box are required for the formation of a high-affinity Transporter-TonB complex. Biochemistry 52: 2638-2648. Freyer, M. W., and E. A. Lewis. 2008. Isothermal Titration Calorimetry: Experimental Design, Data Analysis, and Probing Macromolecule/Ligand Binding and Kinetic Interactions. In Methods in Cell Biology, Correia, J. J.; Detrich III, H. W., Eds.; Academic Press: San Diego, CA, Vol. 84. Ghachi, M. E., Bouhss, A., Barreteau, H., Touz?, T., Auger, G., Blanot, D., and D. Mengin-Lecreulx. 2006. Colicin M Exerts Its Bacteriolytic Effect via Enzymatic Degradation of Undecaprenyl Phosphate-linked Peptidoglycan Precursors. J. Biol. Chem. 281: 22761-22772. Gresock, M. G., Savenkova, M. I., Larsen, R. A., Ollis, A. A., and K. Postle. 2011. Death of the TonB shuttle hypothesis. Front. Microbiol. 2: 206  Grinkova, Y. V., Denisov, I. G., and S. G. Sligar. 2010. Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers. Protein Eng. Des. Select. 23: 843?848. G?nter, K., and V. Braun. 1990. In vivo evidence for FhuA outer membrane receptor interaction with the TonB inner membrane protein of Escherichia coli. FEBS Lett. 274: 85-88. Hagn, F., Etzkorn, M., Raschle, T., and G. Wagner. 2012. Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J. Am. Chem. Soc. 135: 1919-1925. Hancock, R. E. W., and V. Braun. 1976. Nature of the energy requirement for the irreversible adsorption of bacteriophages T1 and ?80 to Escherichia coli. J. Bacteriol. 125: 409-415. Haselwandter, K., and G. Winkelmann. 2002. Ferricrocin - an ectomycorrhizal siderophore of Cenococcum geophilum. Biometals 15(1): 73-77.68 Helbig, S., and V. Braun. 2011. Mapping functional domains of colicin M. J. Bacteriol. 193: 815-821.  Housden, N. G., Loftus, S. R., Moore, G. R., James, R., and C. Kleanthous. 2005. Cell entry mechanism of enzymatic bacterial colicins: Porin recruitment and the thermodynamics of receptor binding. Proc. Natl. Acad. Sci. 102: 13849-13854. Howard, S.P., Herrmann, C., Stratilo, C. W., and V. Braun. 2001. In vivo synthesis of the periplasmic domain of TonB inhibits transport through the FecA and FhuA iron siderophore transporters of Escherichia coli. J. Bacteriol. 183: 5885-5895.	 Hullmann, J., Patzer, S. I., R?mer, C., Hantke, K., and V. Braun. 2008. Periplasmic chaperone FkpA is essential for imported colicin M toxicity. Mol. Microbiol. 69: 926-937. James, K. J., Hancock, M. A., Moreau, V., Molina, F., and J. W. Coulton. 2008. TonB induces conformational changes in surface-exposed loops of FhuA, outer membrane receptor of Escherichia coli. Protein Sci. 17: 1679-1688. James, K. J., Hancock, M. A., Gagnon, J. N., and J. W. Coulton. 2009. TonB interacts with BtuF, the Escherichia coli periplasmic binding protein for cyanocobalamin. Biochemistry 48: 9212?9220. Kadner, R. J., K. Heller, J. W. Coulton, and V. Braun. 1980. Genetic control of hydroxamate-mediated iron uptake in Escherichia coli. J. Bacteriol. 143: 256?264. Kadner, R. J., and K. J. Heller. 1995. Mutual inhibition of cobalamin and siderophore uptake systems suggests their competition for TonB function. J. Bacteriol. 177: 4829-4835. Kampfenkel, K., and V. Braun. 1992. Membrane topology of the Escherichia coli ExbD protein. J. Bacteriol. 174: 5485-5487. Kampfenkel, K., and V. Braun. 1993. Topology of the ExbB protein in the cytoplasmic membrane of Escherichia coli. J. Biol. Chem. 268: 6050-6057. Konisky, J., and B. S. Cowell. 1972. Interaction of colicin Ia with bacterial cells. Direct measurement of Ia?receptor interaction. J. Biol. Chem. 247: 6524-6529. Kurisu, G., Zakharov, S. D., Zhalnina, M. V., Bano, S., Eroukova, V. Y., Rokitskaya, T. I., Antonenko, Y. N., Wiener, M. C., and W. A. Cramer. 2003. The structure of BtuB with bound colicin E3 R-domain implies a translocon. Nat. Struct. Biol. 10: 948-954.69 Khursigara, C. M., De Crescenzo, G., Pawelek, P. D., and J. W. Coulton. 2004. Enhanced binding of TonB to a ligand-loaded outer membrane receptor: role of the oligomeric state of TonB in formation of a functional FhuA-TonB complex. J. Biol. Chem. 279: 7405?7412. Khursigara, C. M., De Crescenzo, G., Pawelek, P. D., and J. W. Coulton. 2005. Deletion of the proline-rich region of TonB disrupts formation of a 2:1 complex with FhuA, an outer membrane receptor of Escherichia coli. Protein Sci. 14: 1266-1273. Khursigara, C. M., De Crescenzo, G., Pawelek, P. D., and J. W. Coulton. 2005. Kinetic analyses reveal multiple steps in forming TonB-FhuA complexes from Escherichia coli. Biochemistry 44: 3441-3453. Killmann, H., Videnov, G., Jung, G., Schwarz, H., and V. Braun. 1995. Identification of Receptor Binding Sites by Competitive Peptide Mapping: Phages T1, T5, and ?80 and Colicin M Bind to the Gating Loop of FhuA. J. Bacteriol. 177(3): 694-698.	 Kim, M., Fanucci, G. E., and D. S. Cafiso. 2007. Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved. Proc. Natl. Acad. Sci. USA 104: 11975-11980. K?edding, J., Howard, P., Kaufmann, L., Polzer, P., Lustig, A., and W. Welte. 2004. Dimerization of TonB is not essential for its binding to the outer membrane siderophore receptor FhuA of Escherichia coli. J. Biol. Chem. 279: 9978-9986.   K?dding, J., Killig, F., Polzer, P., Howard, S. P., Diederichs, K., and W. Welte. 2005. Crystal structure of a 92-residue C-terminal fragment of TonB from Escherichia coli reveals significant conformational changes compared to structures of smaller TonB fragments. J. Biol. Chem. 280:3022-3028. K?hler, S. D., Weber, A., Howard, S.P., Welte, W., and M. Drescher. 2010. The proline-rich domain of TonB possesses an extended polyproline II-like conformation of sufficient length to span the periplasm of Gram-negative bacteria. Protein Sci. 19: 625-630. Krewulak, K. D., and H. J. Vogel. 2008. Structural biology of bacterial irontransport. Biochim. Biophys. Acta. 1778: 1781-1804. Krewulak, K. D., and H. J. Vogel. 2011. TonB or not TonB: is that the question? Biochem. Cell Biol. 89: 87-97.   Larsen R. A., Wood G. E., and K. Postle. 1993. The conserved proline-rich motif is not essential for energy transduction by Escherichia coli TonB protein. Mol. Microbiol. 10: 943?953.70 Larsen, R. A., and K. Postle. 2001. Conserved residues Ser16 and His20 and their relative positioning are essential for TonB activity, cross-linking of TonB with ExbB, and the ability of TonB to respond to proton motive force. J. Biol. Chem. 276: 8111-8117.	 Letain, T.E., and K. Postle. 1997. TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli. Mol. Microbiol. 24: 271-283. Letellier, L., Locher, K.P., Plan?on, L., and J. P. Rosenbusch. 1997. Modeling ligand-gated receptor activity: FhuA-mediated ferrichrome efflux from lipid vesicles triggered by phage T5. J. Biol. Chem. 272: 1448?1451.	 Lef?vre, J., Delepelaire, P., Delepierre, M., and N. Izadi-Pruneyre. 2008. Modulation by substrates of the interaction between the HasR outer membrane receptor and its specific TonB-like protein, HasB. J. Mol. Biol. 378: 840-851. Locher, K. P. and J. P. Rosenbusch. 1997. Oligomeric states and siderophore binding of the ligand-gated FhuA protein that forms channels across Escherichia coli outer membranes. Eur. J. Biochem. 247: 770-775. Locher, K. P., Rees, B., Koebnik, R., Mitschler, A., Moulinier, L., Rosenbusch, J. P., and D. Moras. 1998. Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferricrhome bound states reveal allosteric changes. Cell 95: 771-778.  L?pez, C. S., Peacock, R. S., Crosa, J. H., and H. J. Vogel. 2009. Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillerum. Biochem J. 418: 49-59.  Lukacik, P., Barnard, T. J., Keller, P. W., Chaturvedi, K. S., Seddiki, N., Fairman, J. W., Noinaj, N., Kirby, T. L., Henderson, J. P., Steven, A. C., Hinnebusch, B. J., and S. K. Buchanan. 2012. Structural engineering of a phage lysin that targets Gram-negative pathogens. Proc. Natl. Acad. Sci. 109: 9857-9862. Matzanke, B. F., Bill, E., Trautwein, A. X., and G. Winkelmann. (1988). Ferricrocin functions as the main intracellular iron-storage compound in mycelia of Neurospora crassa. Biometals 1: 18-25.   Messana, I., Angeletti, M., Castagnola, M., De Sanctis, G., Di Stasio, E., Giardina, B., Pucciarelli, S., and M. Coletta. 1998. Thermodynamics of inositol hexabisphosphate interaction with human oxyhemoglobin. J. Biol. Chem. 273:15329?15334.71 Moeck, G.S., Tawa, P., Xiang, H., Ismail, A.A., Turnbull, J.L., and J. W. Coulton. 1996. Ligand-induced conformational change in the ferrichrome-iron receptor of Escherichia coli K-12. Mol Microbiol 22: 459?471. Moeck, G. S., Coulton, J. W., and K. Postle. 1997. Cell envelope signaling in Escherichia coli. J. Biol. Chem. 272: 28391?28397. Moeck, G. S., and  J. W. Coulton. 1998. TonB-dependent iron acquisition: mechanisms of siderophore-mediated active transport. Mol. Microbiol. 28: 675-681. Moeck, G.S., and L. Letellier. 2001. Characterization of In Vitro Interactions between a Truncated TonB Protein from Escherichia coli and the Outer Membrane Receptors FhuA and FepA. J. Bacteriol. 183(9): 2755-2764. Nikaido, H. 2003. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67: 593-656. Noinaj, N., Guillier, M., Barnard, T. J., and S. K. Buchanan. 2010. TonB-dependent transporters: regulation, structure, and function. Annu. Rev. Microbiol. 64: 43-60.   Pawelek, P. D., Croteau, N., Ng-Thow-Hing, C., Khursigara, C. M., Moiseeva, N., Allaire, M., and J. W. Coulton. 2006. Structure of TonB in Complex with FhuA, E. coli Outer Membrane Receptor. Science 312: 1399-1402. Peacock, R., A. M. Weljie, S. P. Howard, F. D. Price, and H. J. Vogel. 2005. The solution structure of the C-terminal domain of TonB and interaction studies with TonB box peptides. J. Mol. Biol. 345: 1185-1197.	 Peacock, R. S., Andrushchenko, V. V., Demcoe, A. R., Gehmlich, M., Lu, L. S., Herrero, A. G., and H. J. Vogel. 2006. Characterization of TonB interactions with the FepA cork domain and FecA N-terminal signaling domain. Biometals 19: 127-142. Pilsl, H., Glaser, C., Gross, P., Killmann, H., ?lschl?ger, T., and V. Braun. 1993. Domains of colicin M involved in uptake and activity. Mol. Gen. Genet. 240: 103-112. Postle, K., and J. T. Skare. 1988. Escherichia coli TonB protein is exported from the cytoplasm without proteolytic cleavage of its amino terminus. J. Biol. Chem. 263: 11000-11007. Postle, K., and R. J. Kadner. 2003. Touch and go: tying TonB to transport. Mol. Microbiol. 49: 869-882. Postle K. 2007. TonB system, in vivo assays and characterization. Methods Enzymol. 422: 245-269.72 Postle, K. and R. A. Larsen. 2007. TonB-dependent energy transduction between outer and cytoplasmic membranes. Biometals 20: 453-465.  Ranquin, A., and P. Van Gelder. 2004. Maltoporin: sugar for physics and biology. Res. Microbiol. 155: 611-616. Ross, P. D., and S. Subramanian. 1981. Thermodynamics of protein association interactions: forces contributing to stability. Biochemistry 20: 3096-3102. Roujeinikova, A. 2008. Crystal structure of the cell wall anchor domain of MotB, a stator component of the bacterial flagellar motor: implications for peptidoglycan recognition. Proc. Natl. Acad. Sci. U.S.A. 105: 10348-10353 Sauter, A., Howard, S.P., and V. Braun. 2003. In vivo evidence for TonB dimerization. J. Bacteriol. 185: 5747-5754. Schaller, K., Dreher, R., and V. Braun. 1981. Structural and functional properties of colicin M. J. Bacteriology 146: 54-63.? Schaller, K., H?ltje, J.-V. and V. Braun. 1982. Colicin M is an inhibitor of murein biosynthesis. J. Bacteriol. 152: 994?1000. Sch?ffler, H., and V. Braun. 1989. Transport across the outer membrane of Escherichia coil K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane. Mol. Gen. Genet. 217: 378-383. Schultz, G., Ullrich, F., Heller, K. J., and V. Braun. 1989. Export and activity of hybrid FhuA'-'Iut receptor proteins and of truncated FhuA' proteins of the outer membrane of Escherichia coil. Mol. Gen. Genet. 216: 230-238.  Shultis, D. D., Purdy, M. D., Banchs, C. N., and M. C. Wiener. 2006. Outer membrane active transport: Structure of the BtuB:TonB complex. Science 312: 1396-1399.  Skare, J. T., Ahmer, B. M. M., Seachord, C. L., Darveau, R. P., and K. Postle.1993. Energy transduction between membranes. TonB, a cytoplasmic mem- brane protein, can be chemically cross-linked in vivo to the outer membrane receptor FepA. J. Biol. Chem. 268: 16302-16308.	 Stefanska, A. L., M. Fulston, C. S. Houge-Frydrych, J. J. Jones, and S. R. Warr. 2000. A potent seryl tRNA synthetase inhibitor SB-217452 isolated from a Streptomycetes species. J. Antibiot. (Tokyo) 53: 1346?1353.73 Thomas, P. G., and J. Seelig. 1993. Binding of the calcium antagonist flunarizine to phosphatidylcholine bilayers: charge effects and thermodynamics. Biochem. J. 291:397?402. Wayne, R., Frick, K., and J.B. Neilands. 1976. Siderophore protection against colicins M, B, V, and Ia in Escherichia coli. J. Bacteriology. 126(1): 7-12.   Weiner, M. C. 2005. TonB-dependent outer membrane transport: going for baroque? Curr. Opin. Struct. Biol. 15: 394-400. Xu, Q., Ellena, J. F., Kim, M., and D. S. Cafiso. 2006. Substrate-Dependent Unfolding of the Energy Coupling Motif of a Membrane Transport Protein Determined by Double Electron-Electron Resonance. Biochemistry 45: 10847-10854. Zeth, K., R?mer, C., Patzer, S.I., and V. Braun. 2008. Crystal Structure of Colicin M, a novel phosphatase specifically imported by Escherichia coli. J. Biol. Chem. 283(7): 25324-25331.74

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0165589/manifest

Comment

Related Items