Rhodopsin is the best characterized member of the large, pharmaceutically important, family of G-protein-coupled receptors (GPCRs), and serves as a prototype for understanding GPCR activation. In this thesis, we aim at understanding the activation mechanism of rhodopsin. To this aim, we first performed an in-depth analysis of the conformational motions of rhodopsin predicted by two elastic network models, Gaussian Network Model (GNM) and Anisotropic Network Model (ANM). We compared these motions with the extensive amount of experimental data, and developed a model for rhodopsin activation. We tested the model with Meta II fluorescence decay rates measured to characterize the deactivation of rhodopsin mutants. We find that our results correctly predict 93% of the experimentally observed effects in 119 rhodopsin mutants for which the decay rates and misfolding data were measured, including a systematic analysis of Cys->Ser replacements. Next, in order to incorporate atomic details and the effects of membrane and water molecules into our model, we developed a new protocol named ANM-restrained molecular dynamics (MD). In this protocol, we used multiple ANM modes as restraints to guide MD simulations. By using this protocol, we were able to sample biologically relevant, large scale motions of the protein that are otherwise not accessible to the conventional timescales MD simulations. Furthermore, we explored the evolution of the multiple ANM global modes with realistic deformations favored by a detailed atomic force field in the presence of the explicit environment. Remarkably, with this method, we identify a highly hinge site, which does not change with several rounds of applying normal modes as restraints. This hinge site includes residues that are directly affected by the isomerization of retinal, as well as those stabilizing the resulting all-trans conformation of the chromophore. The CP ends of the helices H3, H4, H5, and H6 and the connecting loops are found to enjoy an enhanced mobility facilitated by this hinge site. Several new interactions are observed to contribute to the mechanism of signal propagation from the retinal binding pocket to the G-protein binding sites in the CP domain.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04132008-000438 |
| Date | 14 April 2008 |
| Creators | Isin, Basak |
| Contributors | Judith Klein-Seetharaman, Thomas E. Smithgall, Hagai Meirovitch, Mike Cascio, Ivet Bahar |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-04132008-000438/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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