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EXPLORING THE NATURE OF THE TRANSLOCON-ASSISTED
PROTEIN INSERTION
by
Anna Rychkova
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)
August 2013
Copyright 2013 Anna Rychkova
Object Description
| Title | Exploring the nature of the translocon-assisted protein insertion |
| Author | Rychkova, Anna |
| Author email | rychkova@usc.edu;rychkova@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Chemistry |
| School | College of Letters, Arts And Sciences |
| Date defended/completed | 2013-03-14 |
| Date submitted | 2013-07-23 |
| Date approved | 2013-07-23 |
| Restricted until | 2013-07-23 |
| Date published | 2013-07-23 |
| Advisor (committee chair) | Warshel, Arieh |
| Advisor (committee member) |
Haworth, Ian Krylov, Anna |
| Abstract | The main subject of the current dissertation is related to the fundamental question of membrane protein folding. Membrane proteins represent an important class of proteins that is abundant in the most genomes (20-30% of all genes encode membrane proteins) and has significant pharmaceutical interest (target of over 50% of all modern medicinal drugs). Protein misfolding is increasingly recognized as a factor in many diseases, including cystic fibrosis, Parkinson's, Alzheimer's and atherosclerosis. Many proteins involved in misfolding-based pathologies are membrane-associated. Therefore, understanding the mechanism that governs membrane protein folding may aid in curing such diseases. ❧ The vast majority of membrane proteins get inserted into the lipid bilayer through the protein-conducting channel called translocon. The translocon is evolutionary conserved in all kingdoms of life - its homologues are found in eukaryotes, bacteria and archaea. In bacteria the translocon consists of the heterotrimeric SecYEG complex (where ""Sec"" originates from the name of the corresponding gene sec, which stands for secretion). The insertion of proteins into the translocon is performed by a motor protein. For posttranslational protein translocation, the translocon interacts with the cytosolic motor protein SecA that drives the ATP-dependent stepwise translocation of unfolded polypeptides across the membrane. For the cotranslational integration of membrane proteins, the translocon interacts with ribosome-nascent chain complexes and membrane insertion is coupled to polypeptide chain elongation at the ribosome. Together, the complex of SecYEG with the motor protein is termed ""preprotein translocase"" as it suffices for protein translocation. There are some translocase-associated auxiliary proteins that are known to participate in protein insertion into the lipid bilayer by transiently interacting with the translocon (e.g., the heterotrimeric SecD-FyajC complex and the YidC protein). ❧ There are many puzzling questions in the field of membrane proteins. One of such questions involves the discrepancy between the experimental and theoretical estimates for the apparent free energy of inserting charged residues into the lipid bilayer. Here we explore the nature of ΔGapp, asking what should be the value of this parameter if its measurement represents equilibrium conditions. This is done using a coarse grained model with advanced electrostatic treatment. Estimating the energetics of ionized arginine of a transmembrane (TM) helix in the presence of neighboring helixes and/or the translocon provide a rationale for the observed ΔGapp of ionized residues. It is concluded that the apparent insertion free energy of TM with charged residues reflects probably more than just the free energy of moving the isolate single helix from water into the membrane. The present approach should be effective not only in exploring the mechanism of the operation of the translocon but also for studies of other membrane proteins. ❧ The much more advanced question involves the elucidation of the molecular nature of the protein insertion and understanding the mechanisms that govern final membrane protein topology. Here we tried to challenge ourselves and to estimate the complete free energy profile for the translocon-assisted protein translocation and membrane insertion. At present it is not practical to explore the insertion process by brute force simulation approaches due to the extremely long time of this process and the very complex landscape. Thus we use here our previously developed coarse grained (CG) model and explored the energetics of the membrane insertion and translocation paths. The trend in the calculated free energy profiles is verified by evaluating the correlation between the calculated and observed effect of mutations as well as the effect of inverting the signal peptide that reflects the ""positive inside"" rule. Furthermore, the effect of the tentative opening induced by the ribosome is found to reduce the kinetic barrier. Significantly, the trend of the forward and backward energy barriers provides a powerful way of analyzing key energetics information. Thus it is concluded that the insertion process is most likely a non-equilibrium process. Furthermore, we provided a general formulation for the analysis of the elusive apparent membrane insertion energy, ΔGapp, and concluded that this important parameter is unlikely to correspond to the free energy difference between the translocon and membrane. Our formulation seems to resolve the controversy about ΔGapp for Arg. ❧ Having the complete free energy profile for the translocon-assisted membrane protein insertion in hands allowed us to approach an intriguing question about the biphasic pulling force of the translocon. In our current project we are trying to explore the nature of the coupling between the stalling of the elongation of proteins in the ribosome and the insertion through the TR. The origin of this long range coupling is elucidated by coarse grained simulations that combine the TR insertion profile and the effective chemical barrier for the extension of the nascent chain in the ribosome. Our simulation seems to indicate that the coupled TR/chemistry free energy profile accounts for the biphasic force. Apparently, although the overall elongation/insertion process can be depicted as a tug of war between the forces of the TR and the ribosome, it is actually a reflection of the combined free energy landscape. ❧ The results of the current work were published in two PNAS papers: *A. Rychkova, S. Vicatos, A. Warshel, On the energetics of translocon-assisted insertion of charged transmembrane helices into membranes, PNAS, 2010, 107(41), 17598-603; *A. Rychkova, A. Warshel, Exploring the nature of the translocon-assisted protein insertion, PNAS, 2013, 110(2), 495-500, and presented in several conferences, including 2012 Gordon Research Conference ""Protons & Membrane Reactions"", Biophysical Society 55th and 56th Annual Meetings, the 241st ACS National Meeting and the 24th Annual Symposium of the Protein Society. |
| Keyword | apparent free energy; coarse grained model; membrane proteins; signal peptide; topology; translocon |
| Language | English |
| Format (imt) | application/pdf |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Contributing entity | University of Southern California |
| Rights | Rychkova, Anna |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Filename | etd-RychkovaAn-1826.pdf |
| Archival file | uscthesesreloadpub_Volume7/etd-RychkovaAn-1826.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Full text | EXPLORING THE NATURE OF THE TRANSLOCON-ASSISTED PROTEIN INSERTION by Anna Rychkova A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) August 2013 Copyright 2013 Anna Rychkova |
