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Structural and Functional Studies of the Escherichia Coli Yidc

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Structural and Functional Studies of the Escherichia Coli Yidc Structural and functional studies of the Escherichia coli YidC DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Seth William Hennon Ohio State Biochemistry Program The Ohio State University 2015 Dissertation Committee: Professor Ross E. Dalbey, Advisor Professor Thomas Magliery Professor Dehua Pei Professor Natividad Ruiz Copyright by Seth William Hennon 2015 Abstract This dissertation examines the structure and dynamics of the E. coli YidC and explores the membrane insertion of TolQ, which requires YidC and SecYEG. Chapter one of the dissertation is a review of membrane protein targeting and insertion in mainly prokaryotic organisms. There are two bacterial targeting pathways: one pathway is for post- translational translocation and involves a wide variety of components including the chaperones trigger factor, SecA, and SecB; the second pathway is for co-translational targeting and occurs by direct binding of the signal peptide of the ribosome nascent chain complex to the signal recognition particle (SRP) followed by targeting to the membrane and SecYEG. The Sec machinery is conserved in all domains of life and is the major translocase for both co- and post-translational insertion. In its membrane protein insertion function, SecYEG can form a complex with a wide variety of proteins including SecA, the ribosome, SecDF(YajC), FtsY, and the YidC insertase. The structure and function of these proteins are discussed in the first chapter. In addition to exporting proteins by the SecYEG translocase, proteins can be exported by the twin arginine translocation (Tat) pathway. In contrast to the Sec pathway, the Tat pathway acts to transport substrates that exhibit fast folding kinetics, require co-factors, or oligomerize in the cytoplasm before export. Substrates of this pathway have a twin arginine signal peptide which allows targeting to ii the Tat machinery. The Tat complex is composed of two types of proteins: TatC and a combination of TatA like proteins (TatA or TatB). Many characteristics of this pathway are still under debate including: the targeting factors involved, oligomeric state of the Tat complex, and mechanism of pore formation. However, it is known that TatC is the main recognition site for the signal sequence. Chapter two reviews the YidC family of proteins in bacteria, chloroplasts (called Alb3), and the mitochondria (termed Oxa1). All of the homologs contain a core group of five conserved transmembrane (TM) segments while Gram-negative bacteria also contain an extra N-terminal transmembrane segment and a large periplasmic domain. In eukaryotes and Gram-positive bacteria, there are two paralogs: one typically binds directly to the ribosome and functions in co-translational insertion and the other functions in post- translational insertion. A big development in the field during the last year was the determination of two crystal structures of this family of proteins. The structures revealed that YidC contains a hydrophilic cavity that spans the cytoplasmic leaflet of the inner membrane and is exposed to water and a lipid environment. An evolutionarily conserved cytoplasmic hairpin domain was also observed and shown to be dynamic in both structures. Additionally, mechanistic studies have begun to determine the features of a substrate that allow it to be inserted by YidC or by the YidC/Sec complex. In chapter three, in vivo cysteine cross-linking studies were utilized to probe the proximity relationship and dynamics of the five conserved transmembrane domains of the E. coli YidC. Thio-specific homo bi-functional reagents of varying spanner lengths and disulfide iii bond formation catalyzed by iodine were used to probe cysteine pairs located in the membrane border regions or embedded in the membrane. We observed that all of the transmembrane segments probed (TM3, TM4, TM5, and TM6) had a face oriented toward TM2 and that they all came in close contact in the membrane interior. TM2 and TM3 appeared to be in close contact with each other and formed the most cross-links out of all of the cysteine pairs that were tested. The dimeric state of YidC has long been debated but no dimers were observed in our crosslinking studies even with cysteine mutants in both TM2 and TM3 which were proposed to be the interface of a YidC dimer. Our studies also revealed that YidC is a very dynamic protein in the membrane vesicles utilized in our experiments. Both rigid and flexible reagents with a wide range of spanner lengths were able to crosslink most cysteine pairs tested in the cytoplasmic border regions of YidC. This flexibility continued into the membrane interior but disappeared as the cysteine mutants were moved toward the periplasmic half of the membrane. Additionally, the loop that links the large periplasmic P1 domain to TM2 was efficiently crosslinked to the periplasmic portions of TM3 and TM4. This region was previously shown to be important for the function of YidC and we propose that it could act to maintain the permeability barrier when YidC is inserting substrates. These in vivo results support and confirm flexibility that has also been observed in the crystallographic B-factors of the two YidC structures as well as molecular dynamics simulations. They also provide a framework for beginning to tease out the structural dynamics and conformational changes that occur during the catalytic cycle of YidC. However, many of the proximity relations between the TM segments of YidC determined by crosslinking did not fit with the reported crystal structures. The large iv discrepancies between the crosslinking and x-ray structures could be due to the fact that YidC is a very dynamic protein, interacts with many proteins in vivo, or possibly due to some misfolded YidC in our studies. Finally, chapter 4 investigates the membrane insertion of the E. coli TolQ protein (part of the Tol-Pal complex) which was previously thought to insert into the membrane in a Sec independent manner. This protein, containing a short (19 residue) periplasmic N-tail and a short (18 residue) periplasmic loop, was chosen as a potential substrate of YidC because it has features of known YidC substrates and also because it was proposed to insert by a Sec-independent mechanism, which suggest that YidC may be involved. By studying the wild-type TolQ protein, we determined that both YidC and the Sec pathway were able to facilitate insertion of the TolQ periplasmic loop and that SecA and the proton motive force (pmf) were not required. We also made mutations in the periplasmic loop of TolQ to determine which structural properties make a protein dependent of YidC and the Sec translocase. Interestingly, mutations of the loop, which normally contains one positively charged glutamic acid and one negatively charged lysine, altered the requirements. Increasing the number of charged residues and thus the overall hydrophilicity of the loop caused an increased dependence on YidC and SecYEG and adding two negatively charged residues caused insertion to be completely dependent on both insertases. Based on these results, we hypothesize that the P1 loop of TolQ inserts at the interface of YidC and SecYEG in the holo-translocon but further studies are needed in order to provide a comprehensive understanding of the structural features that dictate the translocase requirements for membrane insertion. v Dedication This document is dedicated to my family. vi Acknowledgments First, I would like to thank my advisor, Dr. Ross E. Dalbey, for his guidance and critical analysis of my data as well as his encouragement and support throughout the course of my graduate studies. Additionally, I am also grateful for the constructive criticism and helpful suggestions that were provided by Dr. Stephen White and Dr. Andreas Kuhn regarding my cross-linking project. I would like to thank all of my committee members Dr. Thomas Magliery, Dr. Natividad Ruiz, and Dr. Dehua Pei for their time and guidance. I would also like to thank former and current lab members Dr. Peng Wang, Dr. Jijun Yuan, Dr. Lu Zhu, Dr. Raunak Soman, Yuanyuan Chen, Bala Subramani, Karthika Shanmugam, and Haoze He for their advice and friendship. Most importantly, I would like to thank my parents, Pam and Dave Hennon; my sister, Amanda Hennon; and my family members for their love and support. Lastly, I would like to thank my friends for their encouragement and support during this journey. vii Vita October 6, 1984 ..............................................Born in Warren, OH 2003................................................................Joseph Badger High School 2007................................................................B.S. Biology and Chemistry, Mount Union College 2007 - Present ...............................................Graduate Teaching and Research Associate, Department of Chemistry and Biochemistry, The Ohio State University Publications Hennon, S.W., Soman, R., Zhu, L., and Dalbey, R.E. (2015) YidC/Alb3/Oxa1 family of insertases. Journal of Biological Chemistry. Accepted; waiting to be published as a group with other articles. Hennon, S.W., and Dalbey, R.E. (2014) Cross-linking-based flexibility and proximity relationships between the TM segments of the Escherichia coli YidC. Biochemistry. 53, 3278-3286, doi: 10.1021/bi500257u. viii Fields of Study Major Field: Ohio State Biochemistry Program ix Table of Contents Contents Abstract .............................................................................................................................
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