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Characterization of the interaction between E. coli FtsY and the SecYEG complex Chan, Kenneth Ken-Yin 2007

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Characterization of the Interaction between E. coli FtsY and the SecYEG complex by Kenneth Ken-Yin Chan B.Sc, The University of British Columbia, 2002 B.Ed., The University of British Columbia, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA January 2007 © Kenneth Ken-Yin Chan, 2007 Abstract Translocation of proteins into or across the lipid bilayer is an essential process for all living cells. The Sec translocon, known as Sec61ayP in eukaryotes and SecYEG in prokaryotes, is composed of three subunits in which the largest one, Sec61cc or SecY, constitutes the protein channel or pore in the membrane. For the majority of integral membrane proteins, translocation is performed through the signal recognition particle (SRP) pathway. In this pathway, the SRP recognizes and binds to the leading signal sequence of nascent proteins emerging from translating ribosomal units. Through an interaction with an SRP receptor (SR), located at the membrane, the SRP-ribosome-nascent chain complex is brought to close proximity of the Sec complex. In this thesis, the interaction between the prokaryotic SRP receptor, FtsY, and the SecYEG complex is analyzed. The A domain of FtsY is identified as the SecYEG-FtsY . interacting domain by Blue Native PAGE and analytical gel filtration. Using surface plasmon resonance technique, the binding affinity of FtsY to the Sec translocon is measured. Finally, the interaction seems to inhibit the GTPase activity of FtsY. ii T A B L E O F C O N T E N T S Abstract ii Table of Contents ....iii List of Figures vii List of Abbreviations ix Acknowledgements x Dedication xi CHAPTER ONE: INTRODUCTION... 1 1.1 Preface '. 1 1.2 Secretion: Protein Translocation from Cells 2 1.3 Secretion Systems of Gram-negative Bacteria 3 1.3.1 Type I Secretion System 3 1.3.2 Type II Secretion System 4 1.3.3 Type III Secretion System 4 1.3.4 Type IV Secretion System .4 1.3.5 Type V Secretion System 5 1.3.6 Other transporter systems 5 1.4 Importance of the Sec System in Protein Secretion 6 1.5 Background on Sec Translocon 8 1.6 Protein translocation via the SecA/SecB system i 10 1.7 Protein translocation via the signal recognition particle pathway 12 1.7.1. Signal sequences in protein targeting 12 1.7.2. Components of SRP-mediated protein translocation 12 1.8 The bacterial SRP components: 4.5S RNA and Ffh.... 16 1.9 The bacterial SRP receptor protein, FtsY 17 1.10 The FtsY/Ffh Heterodimer ,.' 19 1.11 GTPase activity of Ffh and FtsY 21 1.12 Thesis Investigation 21 iii CHAPTER TWO: EXPERIMENTAL PROCEDURES 23 2.1 Materials , 23 2.2 Methods '. 24 2.2.1. Cloning and Strains 24 2.2.1.1. Polymerase Chain Reaction Conditions 24 2.2.1.2. Purification of Amplified Products from PCR 24 2.2.1.3. Plasmid and DNA fragment Digestion by Restriction Enzymes 25 2.2.1.4. DNA Ligation Reactions 25 2.2.1.5. Sec Constructs 25 2.2.2. Sequencing at UBC's Nucleic Acid Protein Service Unit (NAPS) 25 2.2.3. Making Competent Cells 26 2.2.4. Transformation of E. coli Strains 26 2.2.5. Expression of Protein 27 2.2.6. Cell Disruption and Protein Purification 27 2.2.6.1. Fractionation of Cytosolic Proteins using MonoQ Resin 27 2.2.6.2. Purification of FtsY Proteins 28 2.2.6.3. Protein Purification of Sec Complexes 28 2.2.7. Sodium Dodecyl Sulphate Polyacyrlamide Gel Electrophoresis (SDS-PAGE)...... 28 2.2.8. Radiolabeling Proteins 29 2.2.9. Blue Native-PAGE and Autoradiography 29 2.2.10. Analytical Gel Filtration Experiments'. 29 2.2.11. Membrane Vesicle Preparations 30 2.2.11.1. Membrane Isolation 30 2.2.11.2. Purification of Inner Membrane Vesicles using Sucrose Density Gradient Centrifugation 30 2.2.11.3 Strip IMVs of Peripherally Associated Proteins 30 2.2.12. Specific Binding Analysis using Unradiolabeled Competitor 30 2.2.13. Surface Plasmon Resonance Experiments 31 2.2.14. GTPase Assay 32 iv 2.2.15. Sample Preparation for Electrospray MS/MS 32 2.2.15.1. Sample Gel Electrophoresis 32 2.2.15.2. Reducing, Alkylating, and Trypsin Digest 33 2.2.15.3. Extraction of Digested Protein 33 2.2.15.4 Electrospray Ionization Quad TOF MS/MS protein identification 33 CHAPTER THREE: RESULTS AND DISCUSSION 35 3.1. Isolation of the FtsY protein 35 3.2. Blue Native Polyacrylamide Gel Electrophoresis Analysis of FtsY with the SecYEG complex 37 3.2.1. The Blue Native polyacrylamide gel electrophoresis technique 37 3.2.2. The SecY E D PEG construct 37 3.2.3. Analysis of FtsY with the Sec Complex 39 3.3. Purification and characteristics of cloned products 40 3.4. BN-PAGE anaylsis with the his-tagged clones 42 3.5. Gel Filtration analysis of FtsY binding to the Sec complex 45 3.6. Determining the importance of the N-terminus of FtsY in the association to the Sec complex using gel filtration 48 3.7. Using the fused Sec dimer in Gel Filtration Analyses with FtsY 50 3.8. Gel Filtration Analysis of FtsY domain mutants 51 3.9. Step-wise Increase of FtsY protein in Analytical Gel Filtration 58 3.10. Identification of the unknown protein complexing with SecYEG 62 3.10.1. BN-PAGE of MonoQ fractions with SecY E D PEG 62 3.10.2. Mass spectrometry of unknown protein 62 3.10.3. EF-Tuongel filtration 64 3.11. Studying the interaction without solubilization of SecYEG : 66 3.11.1. Specific Binding Analysis for FtsY and NG domain on SecYEG membranes 66 3.11.2. Using Surface Plasmon Resonance to test for FtsY-SecYEG interaction 69 3.11.2.1. Surface Plasmon Resonance Background 69 3.11.2.2. SPR with SecA and FtsY 69 3.11.2.3. Affinity of FtsY for SecYEG 73 3.12. Effect of SecYEG on FtsY GTPase Activity 75 CHAPTER FOUR: CONCLUSIONS 78 REFERENCES 81 vi List of Figures: Figure 1.1 Protein Secretion Pathways in Gram-negative bacteria 7 Figure 1.2 Structure of the SecYEG translocon 9 Figure 1.3 Model of Translocation involving SecYEG and SecA 11 Figure 1.4 The Signal Recognition Particle 14 Figure 1.5 Schematic illustration of the SRP pathway for targeting of proteins to the membrane 15 Figure 1.6 FtsY domains and their respective theoretical isoelectric values ..' 18 Figure 1.7 The crystal structure of the Ffh and FtsY NG domain complex 20 Figure 3.1 Isolation of the FtsY protein by MonoQ ion exchange chromatography 36 Figure 3.2 Detection of a FtsY-SecYEG complex using BN-PAGE 38 Figure 3.3 Construction and Purification of truncated FtsY mutants 41 Figure 3.4 Detection of complexes between SecY E D PEG and FtsY mutants using BN-PAGE analysis 43 Figure 3.5 Analysis of the FtsY-SecYE D PEG complex by gel filtration 46 Figure 3.6 SDS-PAGE Analysis of the Protein Fractions obtained by gel filtration 47 Figure 3.7 Analytical gel filtration of FtsY mutants in the presence and absence of SecY E D PEG 49 Figure 3.8 Analytical gel filtration of FtsY with SecEYYG 52 Figure 3.9 Analytical gel filtration of FtsY-A mixed with SecY E D PEG 53 Figure 3.10 Analytical gel filtration of FtsY-A mixed with SecEYYG 55 Figure 3.11 Analytical gel filtration of FtsY-NG mixed with SecY E D PEG 56 Figure 3.12 Analytical gel filtration of FtsY-NG mixed with SecEYYG 57 Figure 3.13 Analytical gel filtration of varied amount of FtsY with constant concentration of purified Sec complex 59 Figure 3.14 Analytical gel filtration of varied amount of FtsY-A with constant concentration of purified Sec complex ". 60 Figure 3.15 Analytical gel filtration of varied amount of Fts Y-NG with constant concentration of purified Sec complex 61 Figure 3.16 Detection of an unknown protein forming a complex with SecYEG using BN-PAGE analysis 63 Vll Figure 3.17 Analytical gel filtration of EF-Tu 65 Figure 3.18 Specific Binding assays using unradiolabeled competitor for FtsY and FtsY-NG 68 Figure 3.19 Surface plasmon resonance sensogram for different concentrations of SecA analyte 70 Figure 3.20 The SPR response curve plot for SecA 71 Figure 3.21 Surface plasmon resonance sensogram for different concentrations of FtsY analyte 72 Figure 3.22 The SPR response curve plot for FtsY 74 Figure 3.23 Inhibition of FtsY and FtsY-NG GTPase activity due to the presence of purified SecYEG 77 Vlll List of Abbreviations: A19 deletion of 19 amino acids A58 deletion of 58 amino acids A domain acidic rich domain ABC ATP binding cassette ATP adenosine triphosphate BN blue native DDM n-Dodecyl B-D-maltoside DNA deoxyribonucleic acid DTT dithiothreitol EDP SecY E D PEG (a triple mutation of SecY at amino acid positions 357-359 to glutamate, aspartic acid, and proline) EDTA ethylenediaminetetraacetic acid EM electron microscopy ER endoplasmic reticulum EYYG S e c E 2 Y Y G 2 (a gene fusion product of two SecY open reading frames in tandem) Ffh fifty-four homologue GMPPNP guanosine-5 '-[(p,y)-imido]triphosphate GTP guanosine triphosphate 1-125 isotope 125 of iodine IMVs inner membrane vesicles IPTG Isopropyl-B-D-thiogalactopyranosid M domain methionine rich domain MS mass spectrometry NG GTPase domain OAc acetate OD optical density Omp outer membrane protein P-32 isotope 32 of phosphorus PAGE polyacylamide gel electrophoresis PCR polymerase chain reaction PEI polyethylenimine PMF proton motive force RNA ribonucleic acid SDS sodium dodecyl sulfate SPR surface plasmon resonance SR signal recognition particle receptor SRP signal recognition particle TAT twin arginine transporters TBE Tris, Borate, EDTA TM transmembrane TS X G 50 mM Tris-HCl, pH 7.5, X mM NaCl, 10% glycerol TSB transformation storage buffer TTSS type III secretion systems Acknowledgements I would like to thank Professor Franck Duong for this wonderful opportunity and for his generous student researchship during my degree. His helpful suggestions and insightful ideas have led me in the proper direction and has taught me a lot. I would also like to thank Dr. Antoine Maillard, our post doctorate fellow, for making my time both fun and educational. Through his help, I was able to learn many new techniques quickly and correctly. His advice has been extremely crucial in my success. Thanks to our Research Associate Dr. Meriem Alami. Our time was short together, but her input has been very helpful. Thanks to all the past and present undergraduate students, whom have come and gone, for their help and for making the lab more lively and fun. They include Nelson, Patrick, Chasline, Kailun, Jonathan, Shifana, Rui and Nadia. Thanks to Dr. Suzanne Perry and Sherry at the U B C Proteomics Core Facility for performing the mass spectrometry analyses and to Mike Krisinger for his help in performing the SPR experiments at the U B C Biophysics Hub. I express gratitude to my Research Committee, Dr. Rachel Fernandez and Dr. Masayuki Numata, for their advice as well as their criticisms. They have given me focus in times of confusion. Finally, I would like to thank my family and friends for their ever-giving support. Their encouragement has given me the strength and motivation I needed to accomplish my goals. In particular, I thank my wife for always being there. This degree would not have been successful without her. To my wonderful wife xi C H A P T E R O N E I N T R O D U C T I O N 1.1 Preface There are an estimated 1.5 million species of organisms on the planet, of which microscopic organisms make up more than half [1]. The absolute number is difficult to estimate. Suffice to say that Scandinavian researchers found at least 4,000 species of bacteria growing in a single gram of soil [2]. Microbials, which include bacteria, fungi, protozoa, algae, and viruses, are found everywhere; from the air that we breathe to the deepest of ocean trenches. They are thought as the organisms found in earliest part of evolution. They have adapted to survive in many different environments and often grow by the millions [3, 4]. They even dwell within larger, multicellular organisms, sometimes in a symbiotical relationship. When this happens, these microscopic organisms become part of what is known as the normal flora [5]. Sometimes, the relationship between microbe and host is a negative one. In humans, more dangerous microbes are responsible for a large part of diseases that ails the world's populations [6, 7]. Gonorrhea, whooping cough, cholera, anthrax, and tuberculosis are just a few examples of bacterial diseases. These infectious diseases are caused by microscopic organisms that penetrate the body's natural barriers and multiply to create symptoms that can range from mild to deadly. Whether speaking of non-infectious organisms, disease causing bacteria, or just cells in general, the understanding of intracellular and intercellular, mechanisms provide insights to how health can be improved and on life in general. In order for cells to survive, they must have the ability to interact with their environment. Cells must receive external nutrient as well as expel 1 wastes and other proteins and metabolites into their surroundings. This latter process is called secretion, the process of segregating or releasing proteins or chemicals from the cell. 1.2 Secretion: Protein Translocation from Cells The transport of secretory and membrane proteins through or into the phospholipid bilayer is an essential process in all living cells. The process of protein transport occurs at the endoplasmic reticulum of eukaryotic cells [8, 9]. Eukaryotic cells use a highly evolved mechanism to do this. Proteins targeted for exocytosis are synthesized by ribosomes that are docked to the rough endoplasmic reticulum. As they are synthesized, these proteins are simultaneously translocated into the ER lumen [10]. There, they are glycosylated and folded with the aid of molecular chaperones. Misfolded proteins are usually identified here and retrotranslocated to the cytosol, where they are degraded by a proteasome. Those proteins that are properly folded then enter the Golgi apparatus through transport vesicles, where further posttranslational modifications occur. The proteins are then moved into secretory vesicles, traveling along the cytoskeleton, to the edge of the cell. Eventually, the vesicle fuses with the cell membrane and, through exocytosis, the protein contents are released into the cell's environment [11]. Strict biochemical control is maintained over this sequence of events by usage of a pH gradient, an area of intense research. Secretion is not unique to eukaryotes. It occurs in bacteria and archaea as well [12-14]. The process occurs at the cytoplasmic, or inner, membrane. Since these cells do not possess internal membranes, the overall process is not as complex as in eukaryotes. Some translocation systems are common to all the three domains of life [15]. One such example is the ATP binding cassette (ABC) type transporters, a transporter which also functions in pumping essential 2 compounds into the cell [16]. Another conserved secretion system is the Sec pathway, which has homologous counterparts in eukaryotes, archaea, and bacteria [17, 18]. When comparing secretion processes within the bacterial kingdom, Gram-negative bacteria have two membranes, thus making secretion potentially more complex then Gram-positive bacteria. In the case of Gram-negative bacteria, there exist a number of modes or types of secretion. The five major systems are briefly discussed. 1.3 Secretion Systems of Gram-negative Bacteria 1.3.1 Type I Secretion System The type I secretion system is made up of the ATP binding cassette (ABC) transporters [19-21]. ABC transporters are mostly unidirectional. In bacteria, they are predominantly involved in the import of essential compounds that cannot be obtained by diffusion (sugars, vitamins, metal ions, etc.) into the cell. In eukaryotes, most ABC transporters move compounds from the cytoplasm to the outside of the cell or into an intracellular compartment, or organelle. In this type I system the substrate is secreted directly from the cytoplasm to the environment by specialized machinery consisting of two cytoplasmic membrane proteins, one of which belongs to the family of ABC transporters. As the ABC cassettes hydrolyze ATP, conformational changes occur that are transmitted to the membrane-spanning domains of the transporter. These induced rearrangements translocate the substrate from one side of the membrane to the other. In Gram-negative bacteria, an additional outer membrane protein is required to allow proteins to pass the second membrane layer. Molecules transported using the type I systems lack classical signal sequences [22]. 1.3.2 Type II Secretion System The type II secretion system is a Sec-dependent pathway that occurs in two steps. In the first step, the proteins are translocated across the cytoplasmic membrane by the general Sec export pathway. The proteins have a classical signal sequence that is recognized and needed for targeting to the translocon. The proteins then have to pass the outer membrane in a second step. The secretory proteins in the outer membrane involved in this step may vary depending on the protein substrate [23]. Due to the known number of Sec-dependent preproteins, the type II secretion system is thought to be the major export pathway in most gram-negative organisms. 1.3.3 Type III Secretion System Type III secretion systems (TTSS) are homologous to the structure and mechanism of the bacterial flagellar basal body and are referred to as contact-mediated secretion systems [24, 25]. It is like a molecular syringe through which a bacterium can inject proteins into eukaryotic cells. Much detail about the molecular mechanisms of the type III export pathway remains elusive to researchers. These mechanisms involve a whole set of proteins that are distinct from the previous two types of secretory pathways. Approximately 20 linked genes are necessary to achieve functional secretion [26]. 1.3.4 Type IV Secretion System The type IV secretion system is homologous to conjugation machinery of bacteria as well as the machinery of the archaeal flagella. It is capable of transporting both DNA and proteins. It was discovered in Agrobacterium tumefaciens, which uses this system to introduce the Ti plasmid and proteins into the host [27]. 4 1.3.5 Type V Secretion System Type V secretion systems are also called the autotransporter system [28]. Like the type II secretion system, this mechanism uses a Sec-dependent pathway and cleavage of a classic signal sequence for molecules to translocate across the inner membrane. However, these molecules can direct their own passage across the outer membrane, thus the name autotransporters, apparently by forming a pore through which they pass. They have the capability to form a beta barrel in their C-terminus and insert into the outer membrane to transport the rest of the peptide out. The beta barrel may be cleaved and left behind in the outer membrane [29]. 1.3.6 Other transporter systems Bacteria as well as mitochondria and chloroplasts also use many other special transport systems like the twin arginine transporters (TAT), which may transport proteins without the need of being unfolded unlike the preproteins that utilize the Sec pathway. The name of the system comes from the requirement for two consecutive arginines in the signal sequence required for targeting to this system [29]. Though TAT proteins only transport substrates across the inner membrane of Gram-negative bacteria, it becomes a mode of secretion in the single membrane Gram-positive bacteria. 5 1.4 Importance of the Sec System in Protein Secretion The afore mentioned systems are responsible for much of the translocation that occurs in Gram-negative bacteria [19, 26] (Figure 1.1). From the above descriptions of protein secretion in prokaryotes, the Sec system obviously plays an important role. In fact, the Sec pathway is the main translocation system in many prokaryotes due to the vast number of substrates that require the Sec machinery [30]. Besides being released out of the cell, many proteins are to become integral membrane proteins. A great number of the substrates utilize the Sec system for the purpose of membrane integration. 6 Figure 1.1 - Protein Secretion Pathways in Gram-negative bacteria. Proteins are transported through or into the bacterial envelope via a number of different secretion pathways, of which the first three types are shown here. Type I secretion systems employ an A B C transporter. In Type II secretion, preproteins are first transported into the periplasmic space via the Sec system, and then exited the cell through an outer membrane transporter. In the Type III secretion system, a needle structure is used for direct contact to the host cell enabling protein translocation. The TAT transporter can process proteins with secondary structure. Diagrams taken from [31]. 7 1.5 Background on Sec Translocon The membrane conduit responsible for protein transport is the heterotrimeric complex, called Sec61ayP in eukaryotes and SecYEG in prokaryotes, which is also called the Sec translocon, complex, or translocase. In bacteria, SecY and SecE make up the essential components of the bacterial translocase, where SecY constitutes the channel [32]. Insights on further mechanisms were stemmed from a recent publication of the crystal structure of the Sec complex in Methanococcus jannachii [33, 34] (Figure 1.2A). In Escherichia coli, SecY is comprised of ten transmembrane helices, making up two halves that theoretically separate from each other during lateral release of substrates with membrane spanning regions. The channel is shaped as an hourglass, being narrow at the center (Figure 1.2B). At this center, or pore, is a collection of hydrophobic residues that constitutes a ring structure. This pore ring is proposed to function as a seal, but would widen just enough for the passage of a polypeptide. It may also function in the arrest of preprotein translocation, which occurs prior to lateral release. A short alpha helical structure also extends from SecY and is located within the pore (Figure 1.2A and 1.2B). This structure, called the plug, was recently shown to displace toward SecE during translocation [35, 36]. E. coli and other bacterial species possess a third non-essential subunit, SecG [37]. The SecG subunit stimulates the membrane insertion of the cytosolic molecular motor, SecA, facilitating translocation efficiency [38]. Other components of the Sec pathway exist, namely SecDFyajC [39, 40]. It is thought that this complex domain helps induce translocation arrest, which provides the time needed for translocation intermediates to form. 8 Figure 1.2 - Structure of the SecYEG translocon. B SecE SecY SecG Periplasm S e c Y 1 S< N > SecE Cytosol Structure of the SecYEG translocon determined from X-ray crystallography. (A) Viewed from the cytosolic face, the crystal structure of the SecYEG complex is seen with SecY constituting the channel. The ten transmembrane (TM) domains are split into two halves, the first five (bottom) and the last five (top). At the center, a small alpha-helical structure, called the plug, is present. SecE and SecG are found at the periphery of the channel. (B) A schematic representation of the Sec translocon within the membrane seen from a side view. The cylindrical structure above the pore represents the plug. Crystal structure taken from [34]. 9 Analyses of the SecYEG complex have shown that the protein stoichiometry of the translocon is more complex than simple monomers [41, 42]. The translocation channel exists as dimers and tetramers as well. Low resolution electron microscopy (EM) images of purified mammalian, yeast and bacterial Sec complexes all revealed the translocon exists in a multimeric state [43, 44]. It is unclear why SecYEG complexes exist as oligomers while a single copy of SecYEG seems to form the translocation channel. 1.6 Protein translocation via the SecA/SecB system There are two pathways that direct proteins to the Sec translocon. The first is the SecA/SecB system, which functions in posttranslational chaperoning and mainly for proteins destined for the periplasm, outer membrane, or secretion out to the cell. SecA is a peripheral membrane-associated protein on the cytosolic face, and has high affinity for the SecYEG complex and acidic lipids [45]. Within the cytosol, SecB forms a complex with the mature binding domain of precursor proteins, like proOmpA, the precursor of outer membrane protein (Omp) A [46]. ATP-bound SecA has direct binding affinity to both SecB as well as the leader peptide of precursor proteins [47]. These interactions are necessary for the precursor to be brought to the site of translocation. With preproteins and chaperones set to play, ATP hydrolysis by SecA creates successive insertion steps that drive the transit of the preproteins across the lipid bilayer [48-50] (Figure 1.3). The proton motive force (PMF), a proton gradient across the membrane, is another source of energy that is also needed for this transit [49, 51]. It is uncertain how the PMF supports translocation. It may act on the preprotein in some sort of electrophoretic manner. Alternatively, it may also alter the conformation of the channel allowing subsequent interactions with translocation partners. 10 Figure 1.3 - Model of Translocation involving SecYEG and SecA. A preprotein is brought to the site of translocation with the aid of the SecB chaperone. The preprotein and SecB interact with SecA and the leader peptide is fed into the Sec channel. Through successive rounds of ATP hydrolysis, SecA drives the protein substrate across the channel a few residues at a time. The proton motive force (PMF) plays a role in the translocation process. SecDFyajC also plays a role in the process, but its role is currently unclear. 11 1.7 Protein translocation via the signal recognition particle (SRP) pathway 1.7.1. Signal sequences in protein targeting Much of what is known about signal sequences, or signal peptides, stemmed from the work of von Heijne, who was able to predict where preproteins were targeted by studying the amino acid sequence [53]. The importance of signal sequences was shown in 1999 when Giinter Blobel received the Nobel Prize in physiology for his discovery that proteins have intrinsic signals that govern their transport and localization in the cell. His work showed that defined peptide motifs targeted proteins to their site of function. Most of the proteins targeted for the extracellular space or subcellular locations carry specific signal peptides characterizing the type of secretion or targeting it undergoes. Each signal sequence has a basic N-terminal region (n-region), a central hydrophobic region (h-region), and a more polar C-terminal region (c-region) [54]. Difference in the length of these regions can determine the targeting location for different proteins. In particular, bacteria have evolved to use a very stringent and tight range for the number of residues in the h-region [55]. Bacterial SRPs bind predominately to very hydrophobic signal peptides as opposed to mammalian SRPs, which recognize a wide range of signal sequences [56, 57]. 1.1.2. Components of SRP-mediated protein translocation The second protein-targeting pathway is the universally conserved signal recognition particle (SRP) pathway. The targeting of preproteins to the translocon is mediated by a number of different protein players and the process occurs in a cotranslational manner [14]. In eukaryotic cells, the signal recognition particle targets nascent proteins destined for secretion or membrane insertion [58]. The SRP includes a 7S RNA molecule and six proteins named 12 according to their molecular weights: SRP72, SRP68, SRP54, SRP19, SRP14, and SRP9 [52, 59, 60] (Figure 1.4A). Of these SRP54 plays a definitive role in the targeting process [61, 62]. The targeting reaction involves a series of ordered steps [63] (Figure 1.5). It begins with the SRP binding to the signal sequence of the nascent polypeptide as it emerges from the ribosome. Protein synthesis is temporarily arrested and this ribosome-nascent chain complex is then targeted to the membrane via the interaction between the SRP and the SRP receptor, called SRa. SRa is found mainly on the periphery of the membrane. A membrane-integrated protein, SR[3, acts as the membrane anchor for SRa [52]. Upon arrival at the membrane, the ribosome-nascent chain complex is released onto the translocation apparatus. When bound to the ribosome, the central pore ring of the Sec translocon is lined up with the exit channel of the ribosome where the nascent chain emerges [64]. After the ribosome-nascent chain complex is released, the SRP and SRP receptor dissociate and are recycled into the targeting reaction through a GTPase dependent event [65, 66]. 13 Figure 1.4 - The Signal Recognition Particle. P e r i p l a s m (A) The eukaryotic signal recognition particle (SRP) system. The SRP is made of a 7S RNA component with six bound proteins, named according to their respective molecular weights. In particular, SRP54 interacts with SRa, a component of the SRP receptor (SR), in order for membrane targeting to occur. SRP is the membrane anchor of the SRP receptor. (B) In the prokaryotic SRP system, the SRP is made up of only a 4.5S RNA and the protein Ffh, which interacts with FtsY. The SRa counterpart is found in FtsY, but no homologue exists for SRp. Ffh and FtsY contain and interact through an NG domain, which is responsible for GTPase activity. Diagrams taken from [52]. 14 Figure 1.5 - Schematic illustration of the SRP pathway for targeting of proteins to the membrane. Ribosome SRP binds to the nascent protein as it emerges from the ribosome. The ribosome-nascent chain complex is then targeted to the membrane through the interaction of the SRP and SRP receptor, SRa and SRp\ Subsequently, the ribosome-nascent chain complex is transferred to the Sec61 channel and then the protein is inserted into the membrane of the endoplasmic reticulum (ER). Adapted from [63]. 15 After the major components of the mammalian SRP system were revealed in 1989, it became apparent that the bacterial system processed in a similar, but simpler, way. In bacteria, it is known to mainly operate in the membrane translocation of preproteins destined for membrane integration [52, 67, 68]. The known components of the SRP in E. coli include a 4.5S RNA molecule and Ffh, a homologue of the mammalian SRP54 protein, hence the name fifty-four homologue (Ffh) (Figure 1.4B). At the membrane, FtsY serves as the SRP receptor, resembling the C-terminal end of SRa, the SRP receptor in the eukaryotic system [69]. An important difference is that that prokaryotes lack a homologue to the eukaryotic SRp\ the membrane-integrated protein that anchors the SRP receptor to the membrane. A genomic search for, a homologous counterpart in E. coli failed to show that one such SRP receptor tethering protein existed in bacteria. Some researchers believe that a putative FtsY receptor does exist and that it remains to be discovered [63]. Thus, the mechanism by which FtsY associates at the membrane is still unclear. 1.8 The bacterial SRP components: 4.5S RNA and Ffh The protein Ffh, also known as P48, is comprised of two domains: a positively charged C-terminal M domain (methionine rich domain) and an NG domain, which is responsible for GTPase activity [70, 71]. This GTPase activity is required for the dissociation of the SRP from the ribosome-nascent chain complex and the membrane receptor, FtsY. The 4.5S RNA is 114 nucleotides long and has a specific role in the assembly of the SRP with its receptor [72, 73]. It does this in two ways. Binding of the RNA occurs at the M domain of Ffh [72]. This association stabilizes the M domain and is required for effective modulation of the NG domain [74]. In other words, 4.5S RNA has conformational effects on Ffh. 4.5S RNA also has highly 16 conserved and invariant residues within its terminal tetranucleotide loop and within single-stranded bulge regions that precede the loop [59]. Mutations of these nucleotides have negative effects on the function of the RNA. Changes to the loop strongly influences the SRP-FtsY interaction and mutations in the bulge effect RNA binding to Ffh [75]. 1.9 The bacterial SRP receptor protein, FtsY Originally, FtsY was implied as a cell division protein because its gene is located in the same operon as FtsE and FtsX, proteins involved in cell filamentation [76]. Overexpression of FtsY resulted in the accumulation of particular precursor proteins. Similar observations were made for the overexpression of Ffh or the depletion of 4.5S RNA in earlier experiments [77-79]. Only after these studies were performed was FtsY identified as a component of the SRP pathway. The receptor protein, FtsY, has two domains: a negatively charged N-terminus A domain and an NG domain, which is responsible for GTPase activity, just as in the case for Ffh (Figure 1.6) [80]. The N domain is composed of four alpha helices that are important for conformational control of the G domain [81]. GTPase functionality of the NG domain is disrupted without the N domain [82]. By separating subcellular components using ultracentrifugation methods, both A and NG domains have shown membrane association properties, but the nature of these associations is still largely unknown [81]. FtsY possesses no obvious hydrophobic or membrane spanning regions. It is possible that membrane interactions are mainly electrostatic, since FtsY is highly charged at physiological pH. Data shows an association of FtsY to the polar headgroups of certain lipids in the membrane, namely, phosphatidylethanolamine [83]. By making liposomes with different amounts of various lipids, FtsY was shown to associate preferentially to 17 Figure 1.6 - FtsY domains and their respective theoretical isoelectric values. Isoelectric value (pi): 3.86 5.59 6.23 The FtsY protein is essentially made up of two domains. The N-terminal A domain is made up of 197 amino acids containing many negatively charged residues, which makes the domain very acidic. The C-terminal domain is made up of N and G domains. Together, the NG domain is responsible for the GTPase activity of FtsY. 18 certain lipids and not others. There is also data that shows that short N-terminal deletions disrupt the binding of FtsY to inner membrane vesicles (IMV), but that a G domain deletion is still tolerated [84]. However, when the entire NG domain is deleted, binding is abolished once again. This was shown by vesicle floatation assays, in which FtsY constructs were fractionated with the vesicles if an interaction occurred. Shown by translocation experiments, removal of the A domain of E.coli FtsY renders the protein nonfunctional [85]. Interestingly, one group of researchers claimed that by returning a single phenylalanine residue at the N-terminus end of the NG domain, functionality can be restored [86]. The implications of this finding are still under debate. It is important to note that FtsY only contains the NG domain for some bacterial species [87-90]. Taken together, these results demonstrate that the targeting of FtsY to the membrane is a topic that is still unclear. 1.10 The FtsY/Ffh Heterodimer In recent years, studies have shown that the interactions between Ffh and FtsY to be an evolutionary marvel. In 2004, a crystal structure of a heterodimer of Ffh and FtsY was solved (Figure 1.7) [91]. The interaction is believed to occur between the two homologous NG domains on each protein. This interaction is further supported by a significant interfacing surface, which is three times as large as the well-known Ran and Rap GTPases [92]. It has been shown that, upon binding, the heterodimer undergoes conformational changes [65]. Specific site-mutations of key and conserved residues on the interface surface of either Ffh or FtsY compromise the stability of the heterodimer complex and inhibit certain stages of conformational rearrangements [93]. This shows the importance of these conformational rearrangements for the interaction between Ffh and FtsY. 19 Figure 1.7 - The crystal structure of the Ffh and FtsY NG domain complex. Ffh (left) and FtsY (right) NG domains are shown as ribbons. The two guanidine phosphate nucleotides are shown as space-filled models. The conserved insertion box domain (IBD) loop in both proteins is located above the nucleotides. The IBD is a p-a-p-a domain that is highly conserved in the SRP-type GTPases, but not present in other GTPase families. This crystal structure shows a remarkable symmetric heterodimer stabilized by a large interaction surface. Also, extensive conformational rearrangements are noted within both protein structures when compared to the state of the proteins without bound substrate. Adapted from [92]. 20 1.11 GTPase activity of Ffh and FtsY SRP-mediated protein targeting is tightly regulated by GTP binding and hydrolysis in both the SRP and the SR [52]. Mutations at residues 446 or 449 within the fj domain of FtsY, caused defective translocation and mal SRP binding, meaning that GTP binding is essential for the function of FtsY [94]. The GTP-bound form of FtsY is suggested as its active state [91, 93]. As separate units, the GTPase activities of FtsY and the SRP are basal and considerably low. However, an in vitro experiment that mixed both FtsY and SRP resulted in an activity that was about twelve fold that of Ffh GTPase activity alone [95]. This shows that the two components mutually enhance the activity for each other. 1.12 Thesis Investigation The question of how FtsY is associated to the membrane remains unanswered. Although it has been shown that both A and NG domains may have a role in membrane binding, the true nature of the association is still unclear. FtsY lacks an SR(3 homologue, which could have acted as a membrane anchor. Recently, in 2005, an article was published that showed that FtsY "interacts functionally and physically with the SecYEG translocon" [96]. This group discovered that increasing the concentration of FtsY rescues a SecYEG mutation and restores protein integration of MtlA, an integral membrane protein that is known to utilize the SRP-mediated pathway. By in vitro crosslinking experiments, it was shown that FtsY and SecY come into close proximity to one another, suggesting that an interaction can occur. This notion was further supported by co-elution of SecY with his-tagged FtsY from a nickel affinity column. My thesis addresses two hypotheses: (1) The Sec translocon serves as FtsY's membrane receptor in the bacterial SRP-mediated system, thus eliminating the need of a SR(3 homologue, 21 and (2) not only is the Sec translocon a receptor, but the interaction has an effect on the function of FtsY. The characterization of the binding domain of FtsY to SecYEG will be presented. Further characterization of the interaction involves the determination of affinity strength and the stochiometric binding ratio. Experiments to address how the interaction affects FtsY function will be determined by GTPase assays. 22 CHAPTER TWO EXPERIMENTAL PROCEDURES 2.1 Materials Primer oligos were ordered from Invitrogen. Molecular biology materials were mostly ordered from Qiagen, including MiniPrep Kits, QG buffer, and DyeEx 2.0 Spin Kits. PfUltra polymerase and buffer used in PCR were obtained from Strategene and nucleotides were purchased from Fermentas. DNA sequencing Big Dye and buffer were from Applied Biosciences. Solvents and chemicals were mostly from Fisher, Sigma, Bioshop, or BDH chemicals. IPTG was bought from Invitrogen and DDM was from Anatrace. From ICN, we received DTT and Coomassie dye. Media, LB Broth and Agar (Miller), was obtained from Fisher. All purification and gel filtration columns came from Amersham Biosciences. SDS and related electrophoretic equipment came from BioRad. Radioactive materials, 1-125 and P-32, were from Perkin Elmer Life and Analytical Sciences. PEI-cellulose plates used for thin layer chromatography were purchased from Selecto Scientific. Trypsin, used prior to mass spectrometry, was from Promega. 23 2.2 Methods 2.2.1. Cloning and Strains A pET23 plasmid containing ftsY was already available in the lab. The gene for FtsY was inserted using Ndel and HindlH restriction sites and was not inframe with the six histidine tag. Primers were engineered to pair with the N- and C-terminus of the FtsY open reading frame and to introduce Xhol to the C-terminus. 2.2.1.1. Polymerase Chain Reaction Conditions Polymerase chain reaction (PCR) was made to a final volume of 50 uL using approximately 200 ng of pET23-FtsY as template DNA, 1.2 uL of 10 mM dNTPs, 1.5 uL of 5 uM 5'-Ndel and 3'-Xhol primers, and 1 pL (Units/uL) of PfuUltra polmerase and its corresponding buffer. PCR was performed in a thermomulticycler with the following program: 95°C for 4 min followed with 20 cycles of 95°C for 30 sec, 60°C for 45 sec, and 72°C for 1:45 min. Then the reactions were brought to 72°C for 5 min and held at 8°C. 2.2.1.2. Purification of Amplified Products from PCR PCR products were purified on agarose gels in a 89 mM Tris, 89 mM Borate, 2 mM EDTA (TBE) buffer system. 0.6% agarose was used for fragments longer than 1 kb and 1% agarose for fragments shorter than 1 kb. Bands were cut out under a 302 nm UV transilluminator. The agarose was melted in 600 uL Qiagen QG buffer and incubated at 55°C (about 5 min). DNA was isolated employing Qiagen DNA MiniPrep columns. 24 2.2.1.3. Plasmid and DNA fragment Digestion by Restriction Enzymes PCR products and pET23 plasmid vector were digested in Invitrogen React Buffer 2 (containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, and 50 mM NaCl) using Ndel and Xhol restriction enzymes. Digestion reactions were allowed to incubate for 2 hours at 37°C. Digested products were loaded and run on either 0.6% or 1% agarose gels in a TBE system and re-isolated with Qiagen DNA MiniPrep columns, as before. 2.2.1.4. DNA Ligation Reactions Each fragment was ligated to the digested pET23 vector with the Invitrogen Ligase system at a volume ratio of 2 uL vector to 5 uL insert, which is a molar ratio of approximately 1:3. Reactions were allowed to occur for 2 hrs at 20°C before transformation into competent DH5a cells. 2.2.1.5. Sec Constructs Constructs of SecYEG (wild-type), SecY E D PEG (a triple mutation of SecY at amino acid positions 357-359 to glutamate, aspartic acid, and proline, known as EDP) and S e c E 2 Y Y G 2 (a gene fusion product of two SecY open reading frames in tandem, known as EYYG) were already present in the lab within a pBAD22 plasmid. 2.2.2. Sequencing at UBC's Nucleic Acid Protein Service Unit (NAPS) DNA Clones were prepared for sequencing by performing PCR of a reaction mix containing 150 to 200 ng of plasmid DNA, 2 uL Big Dye Buffer, 4 pL Big Dye Terminator, and 5 uM sequencing primer to a final volume of 20 uL. PCR was performed in a thermomulticycler 25 with the following program: 96°C for 1 min followed with 25 cycles of 96°C for 30 sec, 50°C for 15 sec, and 60°C for 4 min. Then the reactions were brought to and held at 8°C. The 20 p.L sequencing reaction samples were then desalted with terminator dye removal columns by using Qiagen DyeEx 2.0 Spin Kit. 2.2.3. Making Competent Cells Competent cells for transformation were prepared by growing culture in antibiotic-free LB broth until OD 0.6 for DH5a or OD 0.3 for BL21/C43 [97]; Cells were spun down at 3000 g and resuspended in 1/10th volume in ice cold transformation storage buffer (TSB), which is composed of LB broth containing 10% PEG (MW=3350), 5% DMSO, 10 mM MgCl 2 and 10 mM MgS04 .2.2.4. Transformation of E. coli Strains Approximately 50 ng of plasmid DNA was used in each transformation, using 100 uL of competent cells. Mixtures were allowed to stand for 30 min on ice before a 1 min, 42°C heat shock treatment step. Tubes were returned on ice for about 2 min before 500 u.L of LB broth was added. Cultures were then shaken and incubated at 37°C for 1 hour and 100 or 200 u.L samples of culture were plated on LB Agar plus 80 (xg/mL ampicillin plates. 26 2.2.5. Expression of Protein Strains were precultured overnight and started at 1/100 volume in LB broth plus 80 pg/mL ampicillin. Culture was induced at log phase (OD 0.5 to 0.6) and allowed to grow for another 2 to 3 hours. Strains with pET constructs were induced with 1 mM IPTG and pBAD constructs were induced with 0.2% arabinose. 2.2.6. Cell Disruption and Protein Purification Using 1.5 L of culture, expression of protein was performed. Cells were pelleted at 5000g and resuspended in 20 mL T S 3 0 0 G (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% glycerol). Cells were then disrupted with a M-110L Microfluidizer processor from Microfluidics, with 15 passes at 15, 000 psi. Lysates were spun 10 min at 7000g to remove cell debris and unbroken cells, and the supernatant was spun at 55,000 rpm for 45 min at 4°C in a Ti-60 rotor in a Beckman ultracentrifuge to pellet membranes. 2.2.6.1. Fractionation of Cvtosolic Proteins using MonoO Resin Cytosolic extract separated from membranes were loaded and washed on a MonoQ 5/50 GL column with 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 10% glycerol. The bound protein was eluted with a gradual gradient to a buffer containing 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 10% glycerol over 40 min. Flow rate was set at 1.5 mL/min and fractions of 0.5 mL were collected. 27 2.2.6.2. Purification of FtsY Proteins The supernatant fractions were diluted 2 fold with TS 5 0 G and loaded onto a 5mL His Trap column with TS 1 5 0 G. The column was washed with T S 1 5 0 G + 30 mM Imidazole. The column was then re-equilibrated with TS 5 0 G and eluted with TS 5 0 G + 500 mM Imidazole. Using SDS gels to visualize, clean fractions were pooled and reloaded onto a 5 mL HPQ column and washed with TS 5 0 G. Protein was eluted with TS 5 0 0 G. Clean fractions were pooled and concentrated to above 1 mg/mL while bringing salt concentrations to about 100 mM. 2.2.6.3. Protein Purification of Sec Complexes After the ultracentrifugation step during cell disruption, the membranes were resuspended in 10 mL of T S 1 5 0 G and homogenized in an ice cold 15 mL Dounce. Membranes were solubilized in 1% n-Dodecyl B-D-maltoside (DDM) final for 1 hour at 4°C. Samples were loaded onto a 5 mL His Trap column with TS 1 5 0 G + 0.03% DDM. The column was washed with TS 1 5 0 G + 0.03% DDM + 30 mM Imidazole. The column was then re-equilibrated with TS 5 0 G + 0.03% DDM and eluted with TS 5 0 G + 0.03% DDM + 500 mM Imidazole. Further purification was performed using a 5 mL HPSP column. A similar approach was used as before, but with the addition of 0.03% DDM in all buffers. 2.2.7. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 7.5% or 12% SDS polyacylamide gels were used. Sample loading buffer contained 150 mM Tris-HCl, pH 6.8, 10% glycerol, 200 mM p-mercaptoethanol, 2% SDS, and 1.25% bromophenol blue. Gels were stained with a solution containing 10% methanol, 7% acetic acid, and 0.25% Coomassie Brilliant Blue R-250. 28 2.2.8. Radiolabeling Proteins 70 uL of protein sample diluted to 0.3 mg/mL were made with each respective buffer and placed into a tube coated with 50 \xg of l,3,4,6-tetrachloro-3a,6a-diphenylglycouril (Iodogen). The labeling of tyrosine residues was performed by adding 25 uCi of 1-125 to each tube and the reaction was allowed to occur on ice for 10 min [98]. A 20 fold dilution of this reaction was made as the working stock. 2.2.9. Blue Native-PAGE and Autoradiography 4 to 13 % acylamide gels containing 50 mM Bis-Tris, pH 7.0 and 0.2 M caprioic acid were poured with a gradient mixer using 13 x 20 cm glass plates. Gels were run with 50 mM Tricine, 15 mM Bis-Tris, and 0.01% Serva BW Blue Dye in the cathode and 50 mM Bis-Tris in the anode, running at 20 mA for 1:10 hours at 4°C [99]. Gels were fixed and dried. Film was exposed overnight at -80 °C. 2.2.10. Analytical Gel Filtration Experiments Analytical gel filtration experiments were performed using Amersham Superdex 200 10/300 GL columns in 30 mM Tris-acetate, pH 7.5, 150 mM KOAc, 5 % glycerol, and 0.015% DDM or 50 mM Tris-Cl, pH 7.5, 50 mM NaCl, 5 % glycerol, and 0.015% DDM. Protein samples were mixed and incubated at room temperature for 30 min before injection with a 250 uL Hamilton syringe into an AKTA system. Experiments were run at 0.5 mL/min and 200 pL fractions were collected for one column volume of 24 mL. 29 2.2.11. Membrane Vesicle Preparations 2.2.11.1. Membrane Isolation Expression of protein was done with 500 mL of culture. Cells were pelleted at 5000g and resuspended with 3 mL TS 1 5 0 G. Disruptions were performed with French Press in a 4 mL mini pressure cell at 2000 psi. Lysates were spun 15 min at 16,100g (max speed in an Eppendorf bench top microfuge) to remove cell debris and unbroken cells, and the supernatant fluid was spun at 75,000 rpm for 45 min at 4°C in a TLA-110 rotor in table top microultracentrifuge to pellet membranes. 2.2.11.2. Purification of Inner Membrane Vesicles using Sucrose Density Gradient Centrifugation To further purify inner membrane vesicles (IMVs) from crude membranes, two-step discontinuous sucrose gradient ultracentrifugation was used. Crude membrane samples were diluted in 70% sucrose to achieve a 20% final mixture. Each sample was layered onto a 700 uL 50% sucrose fraction, which was layered on top of a 500 pL 70% sucrose fraction. Samples were spun in a TLS-55 rotor at 54, 000 rpm for 4 hours at 4°C in table top microultracentrifuge. IMVs were collected at the 20%/50% interface. The white coloured heavier outer membrane vesicles are located at the 50%/70% interface. 2.2.11.3. Strip IMVs of Peripherally Associated Proteins 1 volume of 50 mM Tris-HCl, pH 7.5, and 10 M Urea was added to about 500 uL of IMVs and was allowed to stand on ice. After 30 min, 1.2 mL of TL buffer (50 mM Tris-HCl, pH 7.9, 50 mM KC1, 5 mM MgCl 2, 1 mM DTT) was added and spun in a TLA-110 rotor at 65, 000 30 rpm for 30 min at 4°C in table top microultracentrifuge. The membrane pellet was rinsed with TL buffer and resuspended in 200 uL of TL buffer. 2.2.12. Specific Binding Analysis using Unradiolabeled Competitor 25 uL of TL buffer containing 5 ug of membrane with and without over expression of SecYEG and 0.4 mg/mL BSA was mixed with 25 uL containing 2 pL of radiolabeled FtsY or NG with different concentrations of their respective unlabeled protein. The series of mixtures were then incubated at room temperature for 15 min. After incubation, 50 pL of TL buffer + 0.2 M sucrose was added, mixed, and spun at 55, 000 rpm in a TLA-55 rotor at 4°C for 15 min. The supernatant fluid was aspirated off and the pellet washed once with 100 uL of TL buffer. Radiation was measured using a Genie gamma counter. 2.2.13. Surface Plasmon Resonance Experiments Surface plasmon resonance (SPR) experiments were performed using a 4 channel Biacore® 3000 SPR system. IMVs in SPR Buffer A (50 mM Tris-HCl, pH 7.5, 50mM KC1, 5 mM MgCh, 1 mM DTT) were passed 15 times through a 100 nm polycarbonate membrane and immobilized on a LI sensor chip. Binding experiments were performed in SPR Buffer B (50 mM Tris-HCl, pH 7.5, 150 mM KC1, 5 mM MgCl2, 1 mM DTT, 0.5 mg/mL BSA) at 25°C. Stability of baseline and regeneration of binding sites were obtained by injection of 100 mM Na2C03, pH 10.8. Data was analyzed using BIAevaluation and Graphpad prism 4.1 computer programs. 31 2.2.14. GTPase Assay Reactions were assembled in 20 uL volume and contained 50 mM HEPES-KOH, pH 7.5, 150 mM KOAc, 1.5 mM Mg(OAc)2, 400 mM NaCl, 0.01% Ci 2 E 9 , and 2 mM DTT. FtsY or NG was added at 5 uM and solubilized SecYEG was added at 12 u.M on ice. To start each reaction, 1 uCi of P -y-GTP was introduced to the mix, and incubated at 25°C or 37°C at the 0 min time point. At different time intervals, 2.5 uL was removed and immediately frozen in liquid N 2 . Reactions were done in triplicate. Upon completion of the assay, samples were thawed on ice and 1 uL volumes were spotted on polyethylenimine (PEI)-cellulose thin layer chromatography plates. Plates were developed in 0.3 M KH2PO4/H3PO4 pH 3.5 and were then dried and exposed to Phosphorlmager to quantitate the amount of GTP hydrolysis. 2.2.15. Sample Preparation for Electrospray MS/MS 2.2.15.1. Sample Gel Electrophoresis Sample containing the protein of interest was run on a clean 12% SDS gel. After decasting, the gel was rinsed for 5 min with 100 mL of water with gentle shaking. This was repeated twice and then the gel was placed into 20 mL of Invitrogen Simply Blue stain for 1 hour at room temperature with gentle shaking. The gel was then washed with water for 1 hour. The protein band was cut out, rinsed in water, and place into an Eppendorf tube. The gel slice was washed with 100 uL of enzyme buffer (50 mM ammonium bicarbonate) and cut into smaller pieces. The sample was centrifuged and all traces of buffer were removed. The pieces were washed twice with 100 uL of acetonitrile, dried under an N 2 stream, and cool on ice for 30 min. 32 2.2.15.2. Reducing, Alkylating, and Trypsin Digest Samples were reduced by adding 75 uL 10 mM DTT in 50 mM ammonium bicarbonate to each and incubated at 56°C for 1 hour. The tubes were centrifuged. and all traces of buffer were removed. 75 uL 55 mM iodoacetamide in 50 mM ammonium bicarbonate was added immediately and allowed to incubate at room temperature for 30 min in the dark. All traces of buffer were removed again and the pieces were washed with 200 uL of enzyme buffer, and washed twice with 100 uL of acetonitrile, removing as much liquid as possible between rinses. Samples were digested with 25 pL of working trypsin solution (5 pL of a 20 ug of trypsin in 100 uL 1 mM HC1 stock diluted with 95 uL 50 mM ammonium bicarbonate) and incubated on ice for 30 min. Excess trypsin solution was then removed and 25 uL of enzyme buffer was added. Samples were allowed to incubate overnight at 37°C. 2.2.15.3. Extraction of Digested Protein Each digested supernatant sample was removed and placed into a clean microtube. The remaining peptide material was extracted with 3 x 10 min incubations of 25 pL extraction solution. Each liquid fraction was added to the digested supernatant sample. Using a CentriVac, the sample was dried until a final volume of 20 uL. The sample was cleaned with a ZipTip, eluted with 80% acetonitrile and the volume reduced to 10 pL under a nitrogen stream. 2.2.15.4 Electrospray Ionization Quad TOF MS/MS protein identification Peptides were identified based on peptide mass fingerprint analyses by use of a PE SCIEX API 300 Triple Quad mass spectrometer (Applied Biosystems). Proteins were identified using the Mascot search engine. Peptides were cross-referenced with a protein database that 33 contained all genus and species, and a database that contained only E. coli proteins. Ions scores were calculated using the formula -10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores greater than 54 indicated identity or extensive homology (p<0.05). Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits. 34 CHAPTER THREE RESULTS AND DISCUSSION 3.1. Isolation of the FtsY protein A plasmid carrying the open reading frame of ftsY controlled under a T7 promoter was previously engineered in the laboratory. The plasmid was transformed into three E. coli strains, BL21, C41, and C43, and tested for the expression of the FtsY protein. The C43 strain gave the best results. C43 is a double mutant host strain that overcomes toxic effects of over expression of membrane proteins in particular [97]. To obtain a larger amount of the FtsY protein, the expression culture was scaled up. The cells were collected by low speed centrifugation. Then the cells were disrupted by a high pressure microfluidizer device. Afterwards, the cytosolic (or soluble) fraction was isolated through various centrifugation steps, including a high speed step to remove any membrane components. This soluble fraction was then applied onto an ion exchange MonoQ column. Proteins that bound to the column were eluted and fractionated by introducing a gradient of high salt. The fractions were then analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 3.1). FtsY is most abundant and is shown as the major component in fraction #21. On SDS-PAGE, FtsY migrates as a bulky protein with characteristic finger-like projections, migrating with the apparent size of about 90 to 95 kDa (fraction #21). The actually molecular weight of FtsY is 54 kDa. In fraction #21, another bulky band is shown to migrate below the upper FtsY- band. This band likely represents N-terminally truncated FtsY of 14 to 19 amino acids residues as reported in literature [77, 84, 100]. It is inferred that this FtsY truncated species was a product of proteolytic cleavage that occurred during the purification steps. This happens regardless of the addition of protease 35 Figure 3.1 - Isolation of the FtsY protein by MonoQ ion exchange chromatography. A cytosolic extract enriched for FtsY was loaded onto MonoQ resin equilibrated in 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 10% glycerol. Proteins were eluted with a linear gradient to buffer containing 50 mM Tris-HCl, pH 7.5, 500 mMNaCl, and 10% glycerol. Flow rate was set at 1.5 mL/min and fractions of 0.5 mL were collected. These samples were resolved on 12% SDS-PAGE gels and stained with Coomassie Blue. Two FtsY-related bands are observed (Lane 21). The upper band is full length FtsY. The lower band is denoted FtsY', a N-terminally truncated species of FtsY. 36 inhibitors during purification. Some also observe a smaller degradation product of size 33 kDa due to a 187 residue cleavage [94]. 3.2. Blue Native Polyacrylamide Gel Electrophoresis Analysis of FtsY with the SecYEG complex 3.2.1. The Blue Native polyacrylamide gel electrophoresis technique Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a technique that is used to analyze protein-protein interactions in non-denaturing conditions [99, 101]. It employs incubating one protein with the radiolabeled form of the other protein. The mixture is then loaded onto a gradient gel and run under native conditions to preserve any complexes that are . formed. A negatively charged blue dye is used to neutralize the positive charges on proteins, - allowing the complexes to enter the gel. The gels are then dried and the protein bands are visualized via autoradiography. Radiolabeled protein can also be loaded in buffer as a control. Visualization of additional bands other than those in the control, called bandshifts, depicts complex formation with the labeled protein. 3.2.2. The SecY E D PEG construct SecYE D PEG, or EDP for short, was the radiolabeled complex used in BN-PAGE. SecYE D PEG is a mutation of three consecutive residues in SecY at residue positions 357 to 359. The three residues arginine-proline-glycine (RPG) were mutated into glutamic acid-aspartic acid-proline (EDP). This mutation is known to have a negative effect on the interaction of SecA with the translocon [102]. In addition, and more relevant to this project, SecY E D PEG was shown to be predominantly a monomer [36] (Figure 3.2, Lane 1 and 11). In solution, SecYEG is normally a dimer. Observing the translocon as a single, monomer band makes the visualization and 37 Figure 3.2 - Detection of a FtsY-SecYEG complex using BN-PAGE. B i FtsY-SecYEG wm} <]—i FtsY'-SecYEG c=D ^ a s i EF -Tu -SecYEG E D P SecYEG dimer SecYEG monomer FtsY: Mono Q Fraction #20 EDP FtsY: Superdex 200 (A) 1-125 radiolabeled SecY E D P EG (EDP) was incubated with serial dilutions of MonoQ fraction #20 and loaded onto a 4-13% native gel (Lanes 2 to 6). Lane A is a sample of fraction #20 resolved on a 12% SDS gel. Lane 1 is radiolabeled SecY E D P EG. (B) FtsY was further purified by gel filtration. Lane B is a sample of the gel filtrated eluate loaded on a 12% SDS gel. Lanes 7 to 10 show the addition of serial diluted amounts of the gel filtrated material. Lane 11 is radiolabeled SecY E D P EG. SecY E D P EG is mostly in the monomer state (dark arrow), although a small fraction is detected as a dimer (white star). Gray arrows indicate bandshifts due to the proteins forming complexes with SecY E D P EG. They are of FtsY and a Sec monomer, FtsY' and Sec monomer, and EF-Tu and Sec monomer as determined in later experiments (see below). Note that only two of the three complex species are observed using gel-filtrated FtsY. 38 interpretation of complexes much easier. For example, if radiolabeled SecYEG were used, two bands would be observed in the control lane. The intensities of these bands would depend on the conditions used, in particular, the amount of detergent and dye. The upper band, the dimer form of the Sec complex, was more intense than the lower monomer band in the conditions used in this thesis. Thus, when incubating a protein sample with SecYEG, it would be uncertain whether a slight increase in intensity of the dimer band was due to small discrepancies between mixes or whether a complex had formed that migrates at the same level. 3.2.3. Analysis of FtsY with the Sec Complex Using BN-PAGE, a number of MonoQ fractions were tested against radiolabeled solubilized SecY E D PEG to see whether a protein complex would form. Three bandshifts shows that SecY E D PEG was indeed forming complexes. To show all three bandshifts, fraction #21 was not used here since one of the lower bands becomes much fainter. Incubating radiolabeled EDP with fraction #20 shows the position of these bands (Figure 3.2A). Orie band migrates above the SecYEG dimer, the second migrates at about the same level as the dimer, and the last band migrates significantly below the dimer band, but remains above the monomer band. In order to discriminate between bands that are due to FtsY and those that are not, fractions containing a significant amount of FtsY were pooled and gel filtrated. A subsequent SDS-PAGE analysis revealed only two protein bands, indicating an increase in purification (Fig 3.2B, Lane B). Then, an additional BN-PAGE experiment using the more pure sample shows only two significant bandshifts. In Lane 7, an additional faint band is also observed above the upper bandshift. The components of this complex are unknown, but most likely contain a dimer of one of the proteins. Since it never appears as a major band, focus was placed onto the two significant bandshifts. In 39 order to determine further whether these significant bands are truly FtsY-related, truncated versions of FtsY created. His-tagged clones of FtsY were engineered and placed into the same pET plasmid under the control of a T7 promoter. Six C-terminally tagged truncation or domain clones were made altogether: full length FtsY, FtsY-A19 an N-terminal truncation of 19 amino acids, FtsY-A58 an N-terminal truncation of 58 amino acids, FtsY-A197 (or FtsY-NG) N-terminal truncation of 197 amino acids leaving just the NG domain, FtsY-AN with only the AN domain, and FtsY-A with only the A domain (Figure 3.3A). 3.3. Pur i f icat ion and characteristics of cloned products The addition of the his-tag did not affect the solubility of the FtsY mutants. The purified set of proteins was all soluble. These proteins confirmed the bulky characteristic of wild-type FtsY, with some mutants migrating with a much larger apparent molecular weight than their actual sizes (Figure 3.3B). In Lane 1, his-tagged full length FtsY appears very similar to its non-tagged purified counterpart. Finger-like projections are observed on SDS-PAGE. By adding in an affinity purification step, made possible by the introduction of the his-tag, purified FtsY was obtained without gel filtration. Also, the protein was more concentrated. For these reasons, a final product was obtained that had lower amounts of the truncated protein created from proteolytic cleavage. A single cleaner band was attained. FtsY-A19 migrated below that of full length FtsY (Lane 2). Its migration was comparable to that of the proteolytic cleavage product. FtsY-A19 was chosen as a construct for this purpose, to confirm that the second band observed after purification was indeed the reported proteolytic cleavage product. FtsY-A58 migrated slightly below FtsY-A19 (not shown). Interestingly, there was a significant drop in apparent migration with FtsY-NG (Lane 3). This large drop cannot be explained with the difference of 40 Figure 3.3 - Construction and Purification of truncated FtsY mutants. (A) Truncation of FtsY mutants were engineered into a pET23 vector. Each protein was his-tagged and expressed in C43 strain. The number of amino acids (#aa) and the calculated molecular weight (MW c ai) for each mutant is indicated. (B) After purification by nickel affinity chromatography, 3 pg of each truncated FtsY mutant was resolved on a 12% SDS gel. Lane 1 is FtsY, lane 2 is FtsY-A19, lane 3 is FtsY-NG, lane 4 is FtsY-AN, and lane 5 is FtsY-A. 41 178 amino acid residues. Fts Y-NG loses its bulkiness as well as the characteristic finger-like projections of FtsY seen on SDS gels. With the deletion of the G-domain instead of the A domain, FtsY-AN recovers the large apparent molecular weight as well as the characteristic projections of FtsY (Lane 4). FtsY-AN and NG have about the same number of amino acid residues, having 282 and 301 residues respectively. Thus, the A-domain is responsible for the characteristic FtsY traits and is the minimal protein sequence that is needed in order to observe them, as seen with FtsY-A. FtsY-A migrates as a less intense band on SDS-PAGE in comparison to the other mutants, which migrate as tight bands (Lane 5). It is possible that the negative charges on the acidic residues are more pronounced without the influence of the neighbouring charged residues in the NG domain, making repulsive forces and charge-charge interactions more crucial. These various constructs were also analyzed by gel filtration (see below, Figure 3.7A) 3.4. B N - P A G E anaylsis w i th the his-tagged clones After purification of the different proteins and re-employing BN-PAGE analysis, the two original bandshifts from gel filtration prove to be FtsY related (Figure 3.4). By analyzing the amount of the shift, one band is deduced to be one monomer of full length FtsY with one monomer of SecYEG and the second band was one monomer of a FtsY-A19-like protein with one monomer of SecYEG. As mentioned before, the species that is similar to FtsY-A19 is mostly likely an unnatural product observed only after the purification process and migrates at the level of the Sec dimer. The band in lane 6 had a comparable intensity to the dimer band in the control lane (Lane 1). Addition experiments confirmed that a bandshift was present. Once again, faint bands were observed above the major bandshifts throughout lanes 3 to 8, but these 42 Figure 3.4 - Detection of complexes between SecY EG and FtsY mutants using BN-PAGE analysis. Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 FtsY FtsY A19 FtsY NG FtsY AN FtsY A 1-125 radiolabeled SecY E D P EG was incubating with 3 uM, 0.75 uM, and 0.18 uM of each FtsY truncated protein. Samples were loaded onto a 4-13% non-denaturing gradient gel and analyzed by BN-PAGE. Lanes 1 and 18 are control lanes with only radiolabeled SecYEG. Upper and lower black arrows indicate the position of SecYEG dimer and monomer, respectively. The addition of FtsY creates a bandshift that migrates above the SecYEG dimer (lane 3 arrow). The addition of FtsY-A19 creates a bandshift that migrates to the same position as the SecYEG dimer (lane 6 arrow). The addition of FtsY-NG creates a bandshift that migrates just above the SecYEG monomer (lane 9 arrow). The addition of FtsY-AN causes aggregation in the sample, but a faint bandshift is detected at higher concentrations of added protein (lane 12 arrow). The addition of FtsY-A creates a very broad signal (lane 15 arrow). 43 bands became less intense with lower amounts of FtsY protein. Like before, focus was placed on the significant bandshifts. The domain mutants were also tested on BN-PAGE for SecYEG interaction. On BN-PAGE, the NG domain forms a complex with SecYEG. However, the binding of the NG domain is weaker than that of full-length FtsY for the solubilized Sec complex. At about 1 uM, FtsY-NG lost its binding for SecYEG while a significant amount of the translocon is still bound to FtsY (Lanes 4 and 10). FtsY-AN and FtsY-A complexes display patterns different from the truncation mutants. Since the sample remained at the top of the gel, FtsY-AN created an aggregation product upon its addition to SecYEG (Lane 12). A faint band in the middle of the lane indicates that perhaps a complex did form, but proved to be an insoluble product. For FtsY-A, a complex has likely formed, but the complex does not seem insoluble in nature. At 3 uM, the A domain forms a strong complex with the translocon, pulling all free SecYEG into a bandshift (Lane 15). However, this band is very large and spans across a significant length of the gel, making molecular weight determination impossible. Thus, the ratio of its components is uncertain. Decreasing the concentration of Fts-A, allows free SecYEG to re-emerge (Lanes 16 and 17). The complex also decreases in size as well as apparent molecular weight. It is hypothesized that changes in the nature of the A domain are occurring due to the interaction. For example, the A domain is being stretched outwards as it associates to more Sec species. The negative charges of the A domain are interacting dynamically with the positive charges of the membrane protein. 44 3.5. Ge l F i l t rat ion analysis of F t s Y b ind ing to the Sec complex To confirm the interaction of the mutants to SecYEG on BN-PAGE and to further characterize FtsY binding, an alternative method to study protein-protein interaction was used. Using analytical gel filtration, solubilized SecY E D PEG and one of the FtsY clones were mixed, incubated at room temperature and applied onto a Superdex 200 column. The Superdex 200 column is the optimal gel filtration column to use when dealing with moieties above 100 kDa. First, FtsY and solubilized SecY E D PEG were mixed and applied to the column. The reason for using SecYE D PEG is the same for in BN-PAGE, and that it removes the dynamic monomer/dimer equilibrium present in wild-type SecYEG. It would also give clues to whether FtsY binding had any dependency on the SecYEG dimer. A number of buffer conditions were attempted to determine which condition provided the best results. The results show what seemed to be an emergence of a larger third peak that eluted prior to either FtsY or SecY E D PEG (Figure 3.5A). However, the increase in the total size of the third peak from the SecY E D PEG was questionable due to small elution volume differences. Fractions were resolved on SDS-PAGE to confirm the components within the third peak, and to determine the formation of a larger complex (Figure 3.6). When FtsY was run alone, it eluted at 12 mL. Similarly, SecY E D PEG eluted at about 12 to 12.5 mL. In a denaturing gel such as SDS-PAGE, protein subunits are dissociated. The Sec complex separates into SecY, the upper band, and SecE and G, the lower band. In a mix and incubated sample, FtsY and SecY E D PEG both eluted earlier when loaded onto the column. FtsY and the Sec translocon subunits also run in quantitative ratios between fractions. These results seemed to indicate complex formation. Since this was the first time this gel filtration column has been used in the laboratory for protein-protein interaction analysis, it was uncertain whether the distances between elution peaks were significant. 45 Figure 3.5 - Analysis of the FtsY-SecY EG complex by gel filtration. B 140 120 « 100 E c o 00 <N 80 8 60 c ra o < 40 20 -20 140 ~ 120 E c o 00 '—• U C ro L. o < 100 80 60 40 20 •20 10 Volume (mL) • F t s Y + E D P FtsY E D P / V""M f / \ y ^— ) 11 i i 13 15 Volume (mL) FtsY + EDP EDP FtsY (A) Analytical gel filtration of FtsY (light gray), S e c Y t u p E G (dark gray), and FtsY incubated with SecY E D P EG (black) and run in 50 mM Tris-Cl, pH 7.5, 50 mM NaCl, 5 % glycerol, and 0.015% n-Dodecyl B-D-maltoside (DDM). (B) Same as in (A) but run in 30 mM Tris-acetate, pH 7.5, 150mM K(OAc), 5 % glycerol, and 0.015% DDM. The gel filtration experiments were performed using Superdex 200 Amersham columns and were run at 0.5 mL/min and collected in 500 pL fractions. 46 Figure 3.6 - SDS-PAGE Analysis of the Protein Fractions obtained by gel filtration. FtsY + EDP M W S 13 13.5 14 ITIITD 13.5 14 14.5 15 9.5 10 10.5 11 11.5 12 12.5 ,3 13.5 14 14.5 1 5 m L ( k D a ) FtsY alone EDP alone The fractions obtained by analytical gel filtration of FtsY, SecY E D P EG, and FtsY incubated with SecY E D P EG (Figure 3.5A) were analyzed by 12% SDS-PAGE. The first two gels correspond to fractions from FtsY alone and SecY E D P EG alone, respectively. The right-most gel corresponds to fractions from FtsY incubated with SecY EG. Note that the elution of both FtsY and SecY E D P EG are shifted to an earlier volume when the two proteins are mixed together. 47 A change in buffer condition from one containing 50 mM NaCl to 150 mM of KOAc gave an interesting result. SecY E D PEG eluted earlier than before and the SecY E D PEG peak moved to the other side of the.FtsY peak (Figure 3.5B). This observation is best explained in that buffer components affected the nature of the charges in the protein and this change in ionic properties in its environment would likely have an effect on it. However, this shift did not solve the original problem of resolution. 3.6. Determining the importance of the N-terminus of FtsY in the association with the Sec complex using gel filtration To obtain clues on whether or not FtsY forms a complex with the translocon, a study with FtsY, FtsY-A19, and FtsY-A58 was done using gel filtration. These experiments would also help determine whether the immediate N-terminus of FtsY was important for complex formation. If one of the truncation clones does not form a complex and FtsY did form a complex in the previous experiments, then data can be attained about the resolution between FtsY and SecYE D PEG species. First, each clone was applied to the column individually to determine elution volumes and was then plotted together for comparison (Figure 3.7A). A similar pattern was observed with regards to size as was seen with SDS-PAGE. A gradual decrease in size is apparent with the truncation clones, FtsY-A19 and FtsY-A58, until a sudden decrease with FtsY-NG, which eluted after 16 mL. The NG domain is much smaller than FtsY-A19 and FtsY-A58. However, Fts Y-NG has an elution pattern that seems to show that it is even smaller than FtsY-A, even though FtsY-A has 104 fewer amino acid residues than Fts Y-NG. This is best explained in that the A domain is bulky and that the NG domain is not. Once again, this supports that the A domain is responsible for the bulky characteristics of FtsY. 48 (A) FtsY mutants (assorted grays) and S e c Y ^ E G (black) were analyzed by gel filtration in 50 mM Tris-Cl, pH 7.5, 50 mMNaCl, 5 % glycerol, and 0.015% DDM. (B) Analytical gel filtration of SecY E D P EG incubated with FtsY (dark gray), with FtsY-A19 (medium gray), with FtsY-A58 (light gray), and with buffer (black). Run in the same condition as in (A). The gel filtration experiments were performed using Superdex 200 Amersham columns and were run at 0.5 mL/min. 49 FtsY, FtsY-A19, and FtsY-A58 mutants were then separately incubated with SecYb U h JEG and were loaded onto the gel filtration column one at a time. In each case, a larger peak was observed that eluted prior to the SecY E D PEG alone peak meaning that each peak was composed of a species with higher molecular weight than SecY E D PEG itself. This supports the possibility that complexes, which are formed with the Sec translocon, elute only 0.5 to 1 mL prior to the components on a Superdex 200 column. If complexes did form, then it also shows that the immediate N-terminus of FtsY is not essential for association to SecYEG. However, it is possible that the interaction occurs due to the global or net charge of the domain and not due to specific residues. Unfortunately, the problem of resolution has not been ruled out. Whether a complex was formed or not is still uncertain. When referring back to Figure 3.5, slight differences in elution volumes are seen between FtsY alone peaks. This was mostly likely due to small changes in the amount of protein added and to the slight decrease in detergent present in the mix. 3.7. Us ing the fused Sec d imer in G e l F i l t ra t ion Analyses wi th F t s Y The fused dimer construct, SecEYYG was used next to determine whether the peaks could be better resolved. SecEYYG, or EYYG for short, is a construct in which two SecY open reading frames are cloned in tandem at the DNA level. The outcome is two attached SecY subunits with their respective translocon partners bound as observed on native PAGE (not shown), which is effectively a fused translocon dimer. Its stoichiometry can be written as E 2 YYG 2 . FtsY was incubated with SecEYYG and applied to gel filtration in a similar way as with SecYE D PEG. Like the gel filtration experiments before, the larger third peak containing the 50 translocon and the peak of translocon alone eluted somewhat close to one another (Figure 3.8A). However, fractions analyzed on SDS shows that the third peak and the FtsY peak were far enough apart that a significant dip in the FtsY component is apparent (Figure 3.8B). This means that there are indeed two populations of FtsY, one in which FtsY is in complex with Sec and one of FtsY alone and unbound. This also means that the previous gel filtration observations were a result of complex formation and that a slight shift is all that is needed to indicate such. Having previously run molecular weight markers through the column, the stoichiometry of the complexes can be inferred. According to the elution volume shift from 10.8 mL with SecEYYG alone to 10.3 mL with FtsY mixed with SecEYYG, it seems that this shift would only correspond to the addition of one FtsY molecule. Thus FtsY and SecEYYG are binding in a 1:1 ratio. 3.8. Gel Filtration Analysis of FtsY domain mutants The interaction of the A and NG domain mutants with SecY E D PEG and SecEYYG were next analyzed on the Superdex 200 column. When applying these protein mutants to the column individually, the A domain had an elution profile comparable to that of FtsY, eluting around the same time as EDP alone. FtsY-NG eluted significantly later than full length wild-type, and SecYE D PEG (Figures 3.9A and 3.11 A). Once again, this reflects that the bulky nature of the FtsY protein was indeed due to the A domain. Since the A domain eluted with about SecYE D PEG, then the emergence of a new peak would not present as much trouble as did the runs with FtsY. A new peak is observed and, as expected, is quite resolved from any of the individual component peaks alone. On SDS gels, the A domain seems to stretch out into earlier fractions meaning that a complex had formed (Figure 3.9B). When mixing FtsY-A with SecEYYG and applying the 51 Figure 3.8 - Analytical gel filtration of FtsY with SecEYYG. 180 160 ] o 140 E c o 00 120 100 u E ro Ju o tf) < 80 60 40 20 0 -20 first peak second peak 10 i i 12 13 — i — 14 15 1*5 Volume (mL) F t s Y E Y Y G F t s Y and E Y Y G B Elution V o l u m e 9 9.5 10 10.5 11 11.5 12 12.5 13 -t Ft sY Y Y E/G (A) Analytical gel filtration of FtsY (light gray), SecEYYG (dark gray), and FtsY incubated with SecEYYG (black). Performed with Superdex 200 column, in 30 mM Tris-acetate, pH 7.5, 150 mMK(OAc), 5% glycerol, and 0.015% DDM. The gel filtration experiments were performed at 0.5 mL/min and fraction size of 200 uL. (B) Fractions from FtsY + SecEYYG gel filtration analyzed on 12% SDS-PAGE. Left gel represents fractions from the first peak and the right gel from the second peak. 52 Figure 3.9 - Analytical gel filtration of FtsY-A mixed with SecY EG 100 80 4 0 20 -EDP A EOP+A B Elution Volume Elution Volume 10.5 11 11.5 12 12.5 13 10.5 11 11.5 12 12.5 13 ,-' ! • - : sSfPP .&Psi8§ «W JPP i FtsY-A •* . M^WNMF HgN^  4| i SecY * - W n.i»il « t H » • SecE/G . . . . (A) Analytical gel filtration of FtsY-A (light gray), SecY E D P EG (dark gray), and FtsY-A incubated with SecY E D P EG (black). Performed with Superdex 200 column, in 50mM Tris-Cl, pH 7.5, 50 mM NaCl, 5 % glycerol, and 0.015% DDM. The gel filtration experiments were performed at 0.5 mL/min and fraction size of 200 uL. (B) Left gel includes fractions from the FtsY-A mixed with SecY E D P EG gel filtration experiment analyzed on 12% SDS-PAGE. Right gel represents fractions from the FtsY-A protein run alone. 53 mix to the column, results were similar to that of Figure 3.8. Two peaks are observed (Figure 3.1 OA). As before, these two peaks were analyzed on SDS-PAGE. A lower percentage gel was used here because FtsY-A migrates very close to YY, the fused dimer Y component of the dimer construct. Some A domain protein is seen to co-elute with EYYG (Figure 3.1 OB). This shows complex formation. When FtsY-NG was incubated with SecY E D PEG and loaded on the gel filtration column, the third peak was not observed. The two peaks that eluted from the column superimposed those of the single peaks when each component was run on their own. Fractions run on SDS-PAGE shows no complex formation (Figure 3.1 IB). Fractions containing SecY E D PEG and fractions containing FtsY-NG are distinct from one another. Employing SecEYYG with FtsY-NG on gel filtration made no difference. No third peak is observed from gel filtration and SDS gels show no fraction with both FtsY-NG and SecEYYG subunits (Figure 3.12A and B). These results strongly support that the A domain of FtsY, and not the NG domain, is responsible for the interaction of the SRP receptor to the translocon. 54 Figure 3.10 - Analytical gel filtration of FtsY-A mixed with SecEYYG B Elution Volume 10 10.6 FtsY-A SecYY SecE/G 11 12 12.6 13 13.2 mL (A) Analytical gel filtration of FtsY-A (light gray), SecEYYG (dark gray), and FtsY-A incubated with SecEYYG (black). Performed with Superdex 200 column, in 30 mM Tris-acetate pH 7.5, 150 mMK(OAc), 5% glycerol, and 0.015% DDM. The gel filtration experiments were performed at 0.5 mL/min and fraction size of 200 u.L. (B) 7.5% SDS gel of fractions from the FtsY-A mixed with SecEYYG experiment. 55 Figure 3.11 - Analytical gel filtration of Fts Y-NG mixed with SecY E D P EG 100 80 E e oo 60 fM CU u c ra •£ 40 o (/> J2 < 20 Jl 10 11 12 13 14 15 16 17 18 Volume (mL) • E D P N G E D P + N G Elution Volume 11 11.5 12 ,15.5 16 16.5 Elution Volume 11 11.5 12 15.5 16 16.5 i FtsY-NG mm) (A) Analytical gel filtration of FtsY-NG (light gray), SecY E D P EG (dark gray), and FtsY-NG incubated with SecY E D P EG (black). Performed with Superdex 200 column, in 50 mM Tris-Cl, pH 7.5, 50 mMNaCl, 5 % glycerol, and 0.015% DDM. The gel filtration experiments were performed at 0.5 mL/min and fraction size of 200 uL. (B) Left gel includes fractions from the FtsY-NG mixed with SecY E D P EG gel filtration experiment analyzed on 12% SDS-PAGE. Right gel represents fractions from FtsY-NG protein run alone. 56 Figure 3.12 - Analytical gel filtration of FtsY-NG mixed with SecEYYG 13 14 Volume (mL) •NG + EYYG -EYYG NG B Elution Volume 10.5 11 16.8 mL SecYY SecE/G FtsY-NG (A) Analytical gel filtration of FtsY-NG (light gray), SecEYYG (dark gray), and FtsY-NG incubated with SecEYYG (black). Performed with Superdex 200 column, in 30 mM Tris-acetate, pH 7.5, 150 mMK(OAc), 5% glycerol, and 0.015% DDM. The gel filtration experiments were performed at 0.5 mL/min and fraction size of 200 uL. (B) 12% SDS gel of fractions from the FtsY-NG mixed with SecEYYG experiment. 57 3.9. Step-wise Increase of F t s Y protein in Analy t ica l Ge l F i l t ra t ion Analytical gel filtration was also used in a series of experiments in which the amount of one component was held constant, while the amount of the other was changed sequentially. Thus, the concentration of SecY E D PEG was held constant at 6.4 uM. As a control, SecY E D PEG was first analyzed in the absence of other proteins (Figure 3.13). With successive runs, more and more FtsY was mixed with the purified translocon and run on gel filtration. The results show that the SecY E D PEG peak slowly decreased in size while a new peak, a moiety larger in molecular weight, slowly emerged and increased in size as more FtsY was present. When FtsY was run alone, its peak eluted after 12 mL, therefore, the chance that the new peak is due to just FtsY alone is eliminated. This shows that a complex is formed and that as more FtsY is used, more complex formation is possible. This technique was repeated using A domain and NG domain mutants (Figures 3.14 and 3.15). In the case of A domain, the same pattern is observed as seen with FtsY. A new peak emerges as more protein is added to the mixture. However, the enlargement of the new peak was not as dramatic as in the case of full-length FtsY. This was due to the fact that the A domain did not possess a large absorbance at 280 nm, unlike FtsY in comparison. Also, FtsY-A is smaller than full length FtsY. In the case of FtsY-NG, once again no complex formation was seen. With increasing amounts of FtsY-NG, only the peak corresponding to the NG domain alone grew in size. These peaks also came at the same elution volume, which occurred about 15.7 mL. No new peak is observed. Furthermore, the peak of SecYE D PEG alone was the same and is superimposable between trials of increasing amounts of FtsY-NG. Once again, gel filtration has demonstrated that the A domain is responsible for the interaction between FtsY with SecYEG. 58 Figure 3.13 - Analytical gel filtration of varied amount of FtsY with constant concentration of purified Sec complex. 120 p.g of FtsY (thick black) and S e c Y t u l J E G (dash black) were run individually. They were mixed to the same concentrations and ran in the same manner (black). In a series of experiments 90 pg, 60 pg, and 30 pg of FtsY was also mixed with SecY E G and applied to gel filtration (assorted grays). Al l runs performed with Superdex 200 column, in 50 mM Tris-Cl, pH 7.5, 50 m M NaCl , 5 % glycerol, and 0.015% D D M . The gel filtration experiments were performed at 0.5 mL/min. 59 Figure 3.14 - Analytical gel filtration of varied amount of FtsY-A with constant concentration of purified Sec complex. 120 pg of FtsY-A (thick black) and S e c Y t u l T i G (dash black) were run individually. They were mixed to the same concentrations and ran in the same manner (black). In a series of experiments 90 pg, 60 pg, and 30 pg of FtsY was also mixed with S e c Y E D P E G and applied to gel filtration (assorted grays). Al l runs performed with Superdex 200 column, in 50 mM Tris-Cl, pH 7.5, 50 m M NaCl , 5 % glycerol, and 0.015% D D M . The gel filtration experiments were performed at 0.5 mL/min. 60 Figure 3.15 - Analytical gel filtration of varied amount of FtsY-NG with constant concentration of purified Sec complex. SecY E G (dark gray) was run alone. The same amount was then mixed with 120 pg of FtsY-NG and ran in the same manner (black). 30 pg of FtsY-NG was also mixed with S e c Y E D P E G and applied to gel filtration (light gray). Al l runs performed with Superdex 200 column, in 50 m M Tris-Cl, pH 7.5, 50 m M N a C l , 5 % glycerol, and 0.015% D D M . The gel filtration experiments were performed at 0.5 mL/min. 61 3.10. Identification of the unknown protein complexing with SecYEG 3.10.1. BN-PAGE of MonoO fractions with SecY E D PEG The next question was to determine what was responsible for the third bandshift in the original BN-PAGE experiment (Figure 3.2A). To answer this question and to also determine if new interacting partners could be found, the cytosolic extract of BL21 cells was isolated and fractionated with an ion exchange MonoQ column. BL21 was used because it is a general expression strain. With radiolabeled SecYE D PEG, a BN-PAGE binding assay was performed to a number of fractions to see if any complexes occurred (Figure 3.16A). Two potential targets were clearly forming complexes with the Sec complex and were seen across consecutive fractions. By matching the Coomassie stained SDS gels with that of the autoradiographic exposures, it seemed that one of these potential proteins was quite abundant in the cytosolic extract and constitutes a major band in a number of consecutive fractions. It was anticipated that more complexes should have been observed with this approach. For instance, SecA, a protein known to bind and interact with SecYEG, should have been found within the fractions eluted from the MonoQ column. SecA is an acidic protein with an isoelectric point of about 5.3 [103]. Why certain proteins failed to show in the fractions is unknown at this time. 3.10.2. Mass spectrometry of unknown protein Mass spectrometry was decided as the best way to identify the major protein of interest that complexed with SecY E D PEG. The protein was isolated from a SDS gel and processed through tryptic digestion. The sample was sent off for Electrospray Ionization MS/MS analysis. Peptide results were cross-referenced to a database of all species as well as one of E. coli only. The concluding results strongly support that the protein is a ribosome interacting partner, 62 Figure 3.16 - Detection of an unknown protein forming a complex with SecYEG using BN-PAGE analysis. EDP #5 #7 #9 #13 #17 #19 #21 #26 EDP Lane 1 2 3 4 5 6 YEG EDP EF-Tu MonoQ #19 (A) BN-PAGE analysis of protein-SecY EG complexes within MonoQ fractions (Figure 3.1). Selected fractions from MonoQ fractionation of cytosolic extract enriched for FtsY were used. Undiluted and three-fold diluted samples from each of the fractions were incubated with 1-125 radiolabeled SecY E D P EG and loaded onto a 4-13% native gel. Top blue arrow indicates position of translocon dimer and the bottom arrow indicates position of the monomer. Upper-right gray arrow indicates position of FtsY-SecY E D PEG complex in fraction #21 lane. Lower-left gray arrow indicates complex of the unknown protein with SecY E D P EG (compare with Figure 3.2A). (B) First lane is radiolabeled SecYEG and second lane is radiolabeled SecY E D P EG. Undiluted and three-fold diluted samples of purified his-tagged EF-Tu (Lanes 3 and 4) and MonoQ fraction #19 (Lanes 5 and 6) were incubated with radiolabeled SecY E D P EG and loaded onto a 4-13% native gel. Top blue arrow indicates position of SecYEG dimer and the bottom arrow indicates position of SecYEG monomer. Gray arrow indicates position of EF-Tu-SecY E D P EG complex. 63 elongation factor Tu (EF-Tu). The role this elongation factor is in protein synthesis. Why this protein would interact with SecYEG is unclear. Furthermore, EF-Tu is a very abundant protein in E. coli cells, thus confirmational data was needed for its interaction with the Sec translocon. Upon viewing the structure, EF-Tu has been noted to also have the NG domain.. It belonged to the same super family as FtsY and Ffh. This led one to surmise that the NG domain may be somewhat responsible for binding to the translocon. To further confirm that it was indeed EF-Tu that was binding on BN-PAGE, a pET plasmid for his-tagged EF-Tu was obtained from Dr. Linda Spremulli of the University of North Carolina. After purification of the expressed EF-Tu protein, BN-PAGE was employed using this clean material. The complex is recovered and confirmed along side the protein in the MonoQ fraction (Figure 3.16B). 3.10.3. EF-Tu on gel filtration To support these findings, EF-Tu was also run on gel filtration. Like in the case with FtsY-NG, no third peak is observed when the protein was run with SecY E D PEG (Figure 3.17). The two peaks of the mixed sample superimposed those that correspond to EF-Tu run alone and SecYE D PEG run alone. Since FtsY-NG and EF-Tu both show complexes with the Sec complex on BN-PAGE and not on gel filtration, there can be a number of possibilities. It seems that some proteins will form complexes in the conditions of BN-PAGE, but do not in solution, like in the case of gel filtration. Figure 3.4 shows an FtsY-NG-SecYE D PEG complex at 3 pM, but no complex is observed in gel filtration with FtsY-NG at 15 pM. Perhaps there is a more encouraging factor. It may be that these outcomes are due to the NG domain. Both proteins belong to the same superfamily of GTPases, both possessing a similar NG domain. It is possible that the interaction of the NG domain to the translocon is true, but that certain aspects of gel filtration prevent the interaction from forming. One possibility is the detergent found in the 64 Figure 3.17 - Analytical gel filtration of EF-Tu E c o OO a> o c ro n i-o n < Volume (mL) EF-Tu EF-Tu + EDP E D P Analytical gel filtration of EF-Tu (light gray), SecY E D P EG (dark gray), and EF-Tu incubated with SecY E D P EG (black). Performed with Superdex 200 column, in 50 mM Tris-Cl, pH 7.5, 50 mMNaCl, 5 % glycerol, and 0.015% DDM. The gel filtration experiments were performed at 0.5 mL/min. 65 buffer. In BN-PAGE, the detergent is likely removed from the mixture within the wells of the gels at the beginning of electrophoresis. However, it is also possible that the nature of BN-PAGE favours artifactual binding. Proteins may experience an increase in local concentration as the samples move down the gradient gel. Nonetheless, it seems that certain traits within EF-Tu or FtsY-NG, whether be the NG domain or not, allows for translocon binding within polyacrylamide gels and native experiment buffer, but not at the gel filtration level. Further experimentation would be required to determine which scenario is the actual case. 3.11. Studying the interaction without solubilization of SecYEG 3.11.1. Specific Binding Analysis for FtsY and NG domain on SecYEG membranes The next step was to perform experiments in which the Sec complex is still within the membrane and not solubilized. It is still uncertain how solubilization affects the nature of the complex. To do this, specific binding analysis using unradiolabeled competitor was used. Radiolabeled FtsY or FtsY-NG was incubated with purified membranes. The idea behind radiolabeling protein is to chemically bind the 1-125 isotope to tyrosine residues within the protein [98]. This reaction occurs on a carbon adjacent to the hydroxyl group carbon in the phenolic structure. Full-length FtsY has two tyrosine residues, both located in the NG domain. For this reason, FtsY-A was unable to be labeled in the same manner as FtsY and FtsY-NG. To a series of these assay tubes, different amounts of cold, unradiolabeled protein was added. These proteins serve as competitors for binding sites on the membrane and as more cold protein is used, less radiolabeled protein can bind. In such a case, the membrane pellet would emit fewer counts than one with fewer competitors present. When comparing between two proteins, the differences in binding affinity and the amount of covalently bond 1-125 per 66 molecule means that a direct comparison between the concentrations of cold material with respect to each protein is not possible. However, if the counts are expressed as percentage of counts when no cold material is present, then comparisons between proteins are possible. In a plot of radiation emission counts versus concentration of cold competitor, FtsY is seen as a downward curve, illustrating that as more cold competitors are present, less labeled protein can bind to the membrane (Figure 3.18). This means that FtsY is binding. It is important to note that when SecYEG is overexpressed in the membrane, the overall number of radiolabeled FtsY binding is greater than that of non overexpressed membranes. This means that there are more sites on the membrane for FtsY binding, namely the enriched portion of SecYEG translocon units. When analyzing FtsY-NG in the same manner as FtsY, membrane with or without overexpression of SecYEG made very little difference to the counts across, the cold competitor concentration range. Two things are noted here. The first is that the lack of a downward curve means that no competition is occurring. In other words, FtsY-NG is not interacting to the membrane in a significant manner. Secondly, little difference between with and without overexpression of SecYEG means that the NG domain is not binding to the SecYEG in the enriched sample. The above means that the NG domain does not bind to the Sec complex, nor does it bind to membrane. 67 Figure 3.18 - Specific Binding assays using unradiolabeled competitor for FtsY and FtsY-NG. 25000 20000 CL £ c 3 O o 15000 10000 5000 500 1000 1500 Concentration of cold competitor (nM) 2000 2500 -FtsY KM9 EYG - FtsY KM9 -NG KM9 EYG - NG KM9 5 pg of membrane with and without over expression of SecYEG were incubated with I-125 radiolabeled FtsY in TL buffer (50 mM Tris-HCl, pH 7.5, 50 mM KC1, 0.4 mg/mL BSA, 1 mM DTT). In a series of assay reactions, increasing amounts of unlabeled protein was added as a competitor. After incubation at room temperature for 15 min, 50 uL of TL buffer + 0.2 M sucrose was added, mixed, and spun at 55, 000 rpm to pellet membranes. The supernatant was aspirated off and the pellet washed once with 100 pL of TL buffer. Radiation was measured using a Genie gamma counter. The same was performed with FtsY-NG. 68 3.11.2. Using Surface Plasmon Resonance to test for FtsY-SecYEG interaction 3.11.2.1. Surface Plasmon Resonance Background The idea behind Surface Plasmon Resonance (SPR) is that membranes are immobilized on a chip surface, having a certain number of binding sites for any analyte that would be passed over it. A laser, on the opposing side of the chip, refracts depending on the amount of mass bound to the analyte side. Therefore, a change in the refractive incident angle gives a quantitative measurement of bound material [104, 105]. In these experiments, like in the case of the specific binding experiments, membranes with and without overexpression of SecYEG were used. By subtracting the non overexpressed from the overexpressed data, information on the binding of analyte to just SecYEG can be obtained. SecA was used as a control since the binding affinity and characteristics of SecA to SecYEG are already well known. Moreover, an article using SPR with SecA binding was recently published [106]. 3.11.2.2. SPR with SecA and FtsY SecA binding to immobilized membranes was performed very successfully (Figure 3.19). Curves are indicative of a fast association rate and a fast dissociation rate. Affinity values that are very comparable to literature values were obtained (Figure 3.20). With the same immobilized membranes, FtsY was passed over the chip in the same manner as SecA. Binding was accurately detected at 0.5 uM (Figure 3.21). It is clear that FtsY had much lower affinity for SecYEG than did SecA, something noted before when performing BN-PAGE experiments (data not shown). However, some binding did occur. It seemed that FtsY had a fast association rate to SecYEG, similar to SecA, but had slow off-rate kinetics. Above 5 pM, it appears that there was an additional binding mechanism taking place. Since the data was corrected for membrane 69 Figure 3.19 - Surface plasmon resonance sensogram for different concentrations of SecA analyte. Membrane preparations enriched and non-enriched for the SecYEG complex were immobilized on a LI sensor chip. SecA was passed over membranes in 50 mM Tris-HCl, pH 7.5, 150 mM KC1, 5 mM MgCb, 1 mM DTT, 0.5 mg/mL BSA at 25°C. Response curves resulting from non-enriched membranes were subtracted from enriched membranes to attain reference corrected response curves seen here. At time 100 s, SecA analyte was passed over the membranes, starting the association phase. After 300 s, only buffer was passed over the membranes, ending the association phase and starting the dissociation phase. The dissociation was given 450 s of buffer flow to reach near completion. 70 Figure 3.20 - The SPR response curve plot for SecA. IMV wtrc Sec A, (ex 1JI5) minus 5uM; data point 100-^ * ^ = 0 . 0 3 2 + / -0 .010 LIM S 75- Rmax ~ 9 5 +/- 8 R U / * R 2 = 0 .975 Q> O 50- f E x p . Date: 7-06-06 CL / used chip <n & 25-J IMV prep: ? / IMV age : 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Sec A (uM) Response units at which SecA reached binding saturation is plotted against SecA concentration. The binding affinity value was derived through computational analysis. Binding isotherm was fitted to a 1 -site binding hyperbola. A dissociation constant (Kd) of 32 ± 10 nM was attained. 71 Figure 3.21 - Surface plasmon resonance sensogram for different concentrations of FtsY analyte. — IMV YEG+++ OuM FTSY wtrc — IMV YEG+++ 0.5uM FTSY wtrc — IMV YEG+++ 1 uM FTSY wtrc IMV YEG+++ 2.5uM FTSY wtrc — IMV YEG+++ 5uM FTSY wtrc IMV YEG+++ 10uM FTSY wtrc — IMV YEG+++ 24uM FTSY wtrc T ' 1 ' 1 r I T I I I ' ! 0 100 200 300 400 500 600 700 800 900 1000 Time « Membrane preparations enriched and non-enriched for the SecYEG complex were immobilized on a LI sensor chip. FtsY was passed over membranes in 50 mM Tris-HCl, pH 7.5, 150 mM KC1, 5 mM MgCb, 1 mM DTT, 0.5 mg/mL BSA at 25°C. Response curves resulting from non-enriched membranes were subtracted from enriched membranes to attain reference corrected response curves. At time 100 s, FtsY analyte was passed over the membranes, starting the association phase. After 300 s, only buffer was passed over the membranes, ending the association phase and starting the dissociation phase. The dissociation was given 450 s of buffer flow to reach near completion. 72 binding, one explanation is that there is initially more binding on membranes overexpressed with SecYEG relative to the wild-type membranes. However, after initial fast binding has finished, the reverse becomes true because SecYEG sites are becoming saturated resulting in a net decrease in slope as seen at 10 pM. Also, at 24 pM, the effect of additional SecYEG sites seems to be quickly overcome by binding to membrane sites as there was very little difference between the two channels. 3.11.2.3. Affinity of FtsY for SecYEG The first affinity value, or dissociation constant, for FtsY for SecYEG was obtained, which is 4.5 ± 1.1 pM (Figure 3.22). This value is still an estimate at the present time. The affinity value was derived from equilibrium binding responses taken at the end of the association phase at 400 s for 0 to 10 pM and the binding isotherm was fitted to a 1 -site binding hyperbola. It would be ideal to repeat the experiment to see if the same results could be found. Also, GMPPNP, a non-hydrolyzable form of GTP, could also be used. Literature shows that the GTP bound form of FtsY is the active form and it is this form that has the potential to bind to the translocon in an efficient manner and to also be ready to bind its targeting counterpart, GTP. bound SRP. The A and NG domain clones were also analyzed on SRP. The data was somewhat inconclusive. With FtsY, the magnitude of the response was already lower than expected. With the domain clones the values dropped even further (data not shown). However, the general trend seemed to support that some binding occurred in the A domain trials while no binding occurred for the NG domain. A clear baseline was observed in NG domain trials. 73 Figure 3.22 - The SPR response curve plot for FtsY, FTSY wtrc, (ex 1__15) 7.5 FTSY (uWf) K d = 4.5+/-1.1 jiM Rmax=103 +/-12RU R 2 = 0.985 Exp. Date: 7-06-06 used chip IMVprep: ? IMVage: 12.5 Response units at which FtsY reached binding saturation is plotted against FtsY concentration. The binding affinity value was derived through computational analysis. Binding isotherm was fitted to a 1-site binding hyperbola. A dissociation constant (Kd) of 4.5 ± 1.1 pM was attained. 74 3.12. Effect of SecYEG on FtsY GTPase Activity In order to study whether SecYEG had an effect on FtsY activity and function, a GTPase assay was performed. Radioactive GTP, which had a Phosphorus-32 isotope at the gamma position, was used to measure hydrolysis of the nucleotide in vitro. Separation of the products was achieved by thin layer chromatography. Samples of the reaction at different time intervals were spotted onto polyethylenimine (PEI)-cellulose plates and developing with acidic phosphate buffer. PEI is a positively charge component that is bound to the cellulose support of the plate. The migration of compounds with more negative charge character would then be more perturbed than those with less negative charge character. This means that inorganic phosphate ions will migrate further than GTP molecules. Incubating the substrate in the presence and absence of FtsY protein, shows that this is indeed true and that very little hydrolysis takes place in the absence of a GTPase as well (Figure- 3.23A). By adding purified SecYEG into the mixture and quantifying the spots with an Image Quant program, comparative GTP hydrolysis was achieved for FtsY and FtsY-NG in the presence and absence of purified SecYEG. The results of the GTPase assays show that FtsY activity was low, which corresponds to findings in the literature. In the presence of solubilized SecYEG, it seems that the activity was even lower, with a decrease of about 5% (Figure 3.23B). Moreover, the activity of NG domain was even lower than that of FtsY. This is a discrepancy with literary findings in which FtsY-NG activity was twice that of FtsY [107]. However, it should be noted that this was only reported from one research group and that the conditions they used were different to the one used here. The GTPase assays performed in this thesis also shows that, in the presence of SecYEG, the activity of FtsY-NG decreased about 10% when compared with the absence of SecYEG. 75 The main question is whether or not these small changes have any biological significance. If the activities of the protein are already low to begin with, small changes to this value would not prove critical and would most probably not cross important, mechanistic thresholds of any sort. In order to rectify this, a larger starting activity value is needed. The introduction of the signal recognition particle could potentially achieve this. According to literature, GTP hydrolysis when SRP and FtsY is both present is about 60 -70 times that of just FtsY alone. However, attempts to detect a greater GTPase activity in the presence of SRP were unsuccessful. Perhaps the detergent component in the buffer is preventing the proper SRP-FtsY interaction, or perhaps the detergent is preventing the reconstitution of the SRP from Ffh and 4.5S RNA. 76 Figure 3.23 - Inhibition of FtsY and FtsY-NG GTPase activity due to the presence of purified SecYEG. Lane 1 Inorganic phosphate GTP B Time(min) 10 20 40 601 |0 20 40 60 | FtsY - + 0.12 0.1 I 0.08 o E D. "S 0.06 N TJ H 0.04 0.02 I GTP FtsY FtsY+Y EG sam pie NG+YEG FtsY or FtsY-NG was incubated in the presence or absence of purified SecYEG for 60 min at 25°C after the addition of P -y-GTP. At the end of incubation, 1 pL samples were spotted on polyethylenimine (PEI)-cellulose thin layer chromatography plates and developed in 0.3 M KH2PO4/H3PO4 pH 3.5. (A) A sample of thin layer chromatography using PEI-cellulose plates. Lanes 1 to 4 correspond to samples of radiolabeled GTP in the absence of FtsY spotted after 0, 20, 40, and 60 minutes of incubation, respectively. Lanes 5 to 8 correspond to samples of radiolabeled GTP in the presence of FtsY in the same time intervals. (B) Spots of remaining GTP were quantified and the amount of GTP hydrolysis determined. CHAPTER FOUR CONCLUSIONS Through BN-PAGE and analytical gel filtration techniques, the interaction between FtsY and solubilized SecYEG were studied. This thesis has served to further support that interaction. These studies have shown that the association is due to the A domain of FtsY and not the NG domain. Furthermore, the interaction it is not dependent on the immediate N-terminus of the A domain in FtsY since truncating the first 58 residues did not abolish Sec binding, whereas deleting 197 residues did. Further support for the association was found with specific binding assays and surface plasmon binding experiments. In the specific binding assays, the difference between binding of FtsY to membranes enriched with SecYEG and to wild-type membranes was apparent., No difference was observed for the NG domain between the two types of membrane. With SPR experiments, the first ever estimate for FtsY affinity for the Sec complex was achieved. This value falls in the single digit uM range. Simple activity studies also showed that SecYEG has an inhibitory effect on FtsY GTPase activity. BN-PAGE has been shown to be a more robust method of showing interaction. It is not certain whether some interactions shown by this method are true or not,- like in the case of EF-Tu. However, it is a good technique for the initial search for protein-protein interaction. This is a clear example where support for findings with different techniques is important. Gel filtration has been a very definitive and reliable method. The results are extremely reproducible given stringency of buffer conditions. In order to further define the characteristics of the protein-protein interaction in question, namely that of FtsY and SecYEG, more can be done with the techniques used in this thesis. The 78 foremost studies should include a GTP bound form of FtsY. It has been suggested that the GTP-bound form of FtsY is its active state and the conformation in which it interacts with SecYEG. The non-hydrolyzable analog of GTP, GMP-PNP, can be used to lock FtsY in a GTP bound state. Preliminary experiments were tried, but much remains to be done. The interaction can also be investigated from the view point of SecY. Cytoplasmic loop deletion mutants of SecY were constructed. The interaction was not hindered within BN-PAGE trials. If BN-PAGE did create artifactual binding, then alternative experiments should be done to either confirm the maintained interaction or to show that certain loop deletions affects FtsY binding. A powerful, yet difficult, crosslinking technique is available that could possibly elucidate regions of binding on both SecY and FtsY simultaneously. In this type of experiment, systematic cysteine mutagenesis across suspected regions of FtsY and SecY would be introduced. After exposing each FtsY/SecY mutant pair to oxidative conditions, the products would be digested and sent for mass spectrometry to identify linked peptides. This would give the exact regions that are in proximity between the proteins. Due to its laborious and problematic nature, it was not attempted in the parameters of this thesis. The laboratory has recently begun studies with nanodiscs, having the Sec complex bound within a lipid bilayer and held together with scaffolding proteins. These experiments could serve to support in-membrane SecYEG experiments, like surface plasmon resonance. It would also provide the ability to perform experiments with membrane bound SecYEG that required solubilized Sec in the past, like in electrophoretic experiments. Loading membranes on native gels would not give conclusive results due to the size limitation of gradient and acrylamide gels. 79 Undoubtedly, these trials would provide data on protein-protein interactions that has SecYEG in more native conformations and would remove regions that were buried in the membrane from question. Finally, the study on the SRP pathway could be extended passed the FtsY-SecYEG interaction. This is the ultimate long-term goal for a project of this nature. 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