Structural and functional studies of the <i>Escherichia coli</i> YidC
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The Ohio State University / OhioLINK
2015
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu14309195832021-08-03T06:31:13Z Structural and functional studies of the <i>Escherichia coli</i> YidC Hennon, Seth William Biochemistry This dissertation examines the structure and dynamics of the <i>E. coli</i> 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 transfer to SecYEG or YidC.Chapter two reviews the YidC family of proteins in bacteria, chloroplasts (called Alb3), and the mitochondria (termed Oxa1). 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. Two recent crystal 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, <i>in vivo</i> cysteine cross-linking studies were utilized to probe the proximity relationship and dynamics of the five conserved transmembrane domains of the <i>E. coli</i> YidC. Thio-specific homo bi-functional reagents of varying spanner lengths and disulfide bond formation catalyzed by iodine were used to probe cysteine pairs located in the membrane border regions or embedded in the membrane. These <i>in vivo</i> 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.Finally, chapter 4 investigates the membrane insertion of the <i>E. coli</i> TolQ protein (part of the Tol-Pal complex) which was previously thought to insert into the membrane in a Sec independent manner. 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. 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. 2015-08-14 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1430919583 http://rave.ohiolink.edu/etdc/view?acc_num=osu1430919583 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
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NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Biochemistry |
| spellingShingle |
Biochemistry Hennon, Seth William Structural and functional studies of the <i>Escherichia coli</i> YidC |
| author |
Hennon, Seth William |
| author_facet |
Hennon, Seth William |
| author_sort |
Hennon, Seth William |
| title |
Structural and functional studies of the <i>Escherichia coli</i> YidC |
| title_short |
Structural and functional studies of the <i>Escherichia coli</i> YidC |
| title_full |
Structural and functional studies of the <i>Escherichia coli</i> YidC |
| title_fullStr |
Structural and functional studies of the <i>Escherichia coli</i> YidC |
| title_full_unstemmed |
Structural and functional studies of the <i>Escherichia coli</i> YidC |
| title_sort |
structural and functional studies of the <i>escherichia coli</i> yidc |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2015 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1430919583 |
| work_keys_str_mv |
AT hennonsethwilliam structuralandfunctionalstudiesoftheiescherichiacoliiyidc |
| _version_ |
1719438205150822400 |