Lipid-Protein Interactions by Advanced EPR Methods

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dc.contributor.advisor Tatyana I. Smirnova, Committee Chair en_US
dc.contributor.advisor Alex I. Smirnov, Committee Member en_US
dc.contributor.advisor Jerry L. Whitten, Committee Member en_US
dc.contributor.advisor Alexander Deiters, Committee Member en_US
dc.contributor.advisor Joseph C. Burns , Committee Member en_US
dc.contributor.author Chadwick, Thomas Gray en_US
dc.date.accessioned 2010-08-19T18:14:12Z
dc.date.available 2010-08-19T18:14:12Z
dc.date.issued 2010-05-27 en_US
dc.identifier.other etd-05082009-112044 en_US
dc.identifier.uri http://www.lib.ncsu.edu/resolver/1840.16/6167
dc.description.abstract Biological membranes contain proteins that are responsible for many vital biological functions, and understanding the structure-function relationships between the two are fundamentally important. In this work we have utilized Electron Paramagnetic Resonance (EPR) spectroscopy to investigate the chemistry of the phospholipid biding protein Sec14p, and the insertion profiles of synthetic peptides corresponding to the E2 transmembrane domain of selected mutants of the Sindbis Virus. Sec14p is associated with the secretory pathway in the Gogli apparatus, and is thought to be involved in regulating membrane composition. It has been shown to bind phosphatidylcholine (PC) and phosphatidylinositol (PI) in vitro. We demonstrate that Sec14p binds spin labeled homologous of the PC lipid, and the corresponding EPR spectra provide information about the mobility of the bound lipid and the polarity within the binding pocket. Accessibility measurements also ascertain the orientation of the PC molecule within the binding pocket. The results show that the PC molecule is highly restricted inside the biding pocket; polarity and procticity decrease with distance from the polar head region, with increase polarity and procticity at the distal end of the sn-2 acyl chain. EPR data indicate that the molecule adopts a headgroup-out orientation and the polarity profile provides a hydrophobic matching necessary for Sec14p to extract a lipid from a membrane in a energy-independent mechanism. Sinbis virus infects both insects and mammal, and replication requires the incorporation of the host cell’s membrane into its structure. The physical properties of these membranes differ, and the transmembrane domains of the virus’s structural proteins must be able to assemble into the same structure in both membrane types. Virus mutants having truncated transmembrane domains exhibit differential ability to reproduce, where certain mutants favor the production of new virus in mammalian cells, while other mutants favor insect cells. We have investigated this differential infectivity of the Sindbis virus mutants by examining the insertion profiles of synthetic peptides STM16 and STM18 in insect and mammalian membrane mimics. These peptides correspond to the transmembrane segments of Sindbis virus mutants TM-16 and TM-18. The results indicate that while both peptides assume transmembrane orientation in the insect membrane mimic, addition of cholesterol affects peptide-membrane interactions in a cholesterol-concentration dependant manner. en_US
dc.rights 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, dis sertation, 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 NC State University 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. en_US
dc.subject spectroscopy en_US
dc.subject electron paramagnetic resonance en_US
dc.title Lipid-Protein Interactions by Advanced EPR Methods en_US
dc.degree.name PhD en_US
dc.degree.level dissertation en_US
dc.degree.discipline Chemistry en_US


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