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dc.contributor.advisorKuczera, Krzysztof
dc.contributor.authorParthasarathy, Sudharsan
dc.date.accessioned2012-10-28T16:36:03Z
dc.date.available2012-10-28T16:36:03Z
dc.date.issued2012-05-31
dc.date.submitted2011
dc.identifier.otherhttp://dissertations.umi.com/ku:11866
dc.identifier.urihttp://hdl.handle.net/1808/10298
dc.description.abstractCytochrome b5 (CYB5) is a small ubiquitous heme binding protein whose biochemical function is electron transfer. Two isoforms of CYB5 are found in mammals, which are named based on their specific subcellular localization. One isoform is localized to the outer membrane of mitochondria (OMb5) and the other isoform is localized to the endoplasmic reticulum (Mcb5). Available evidence indicates that Mc and OM b5 isoforms arose from a gene duplication event that led to functional divergence. High sequence identity is observed when comparing individual isoforms across mammalian organisms. A highly conserved protein fold is also a feature when comparing the two isoforms. However the two isoforms differ remarkably with respect to their biophysical and biochemical properties. This dissertation focusses on analyzing the specific differences in Mc and OMb5 amino acid sequences that translate into subtle differences in structure, and to correlate them with differences in the biophysical properties of the two isoforms. A combination of experimental and theoretical methods has been used in order to study the differences between Mcb5 and Omb5. In chapter 3, I describe the X-ray crystal structures of the rat Omb5 (rOMb5) and human Omb5 (hOMb5), which reveal a striking difference in structure due to a difference in buried residue 21. The presence of Leu at position 21 in hOMb5 causes a displacement of the first two residues in beta 5, and consequent loss of two of the three hydrogen bonds between beta 5 and beta 4. Hydrogen bonding between the residues is instead mediated by two well ordered, fully buried water molecules. MD simulations of the hOMb5 protein, with and without the water molecules, provided mechanistic insight into the similarity in biophysical properties between hOMb5 and rOMb5. This study also provided information on the versatility of the CYB5 fold to resist internal mutations. Chapter 4 is a mutation based study that attempts to combine the most stabilizing features of OMb5 and Mcb5. The model proteins used here are rOMb5 and the bovine Mcb5 (bMcb5). We performed a systematic incorporation of amino acids from rOMb5 into the corresponding positions in bMcb5 which culminated in generating a hybrid protein (hybb5) that combined the most stabilizing features of the two proteins. We were successful in generating a series of mutants of increasing stability that altered both heme binding strength and apo protein stability. Further investigation revealed that rOMb5 displayed greater stability on account of the amino acid at position 71 (Leu). The presence of Leu in OMb5 isoforms results in generation of a more compact empty heme binding pocket that plays a major part in stabilizing OMb5 in contrast to Mcb5 isoforms which contain a more polar residue (Ser) at position 71. Chapter 5 utilizes the mutants generated as part of the study in chapter 4 to understand the factors responsible for modulating the redox potential difference in Omb5 and Mcb5. The initial part of the chapter focusses on measurement of redox potentials of the wild type proteins and the mutants. A constant redox potential difference of 60 mV was observed when comparing Omb5 and Mcb5 proteins in different mammals. Crystal structures of a point mutant of bMcb5 (McS71L) and hybb5 were also generated and studied using molecular dynamics. Analysis of the crystal structures of bMcb5, McS71L, hybb5, rOMb5 and hOM5b revealed an in-plane rotation of the heme of approximately 13°. This in-plane rotation was consistently observed in all the proteins which contained Leu at position 71. The results obtained from experiments in chapter 5 lead us to propose that evolutionary divergence of the two isoforms (Mcb5 and Omb5) was favored by mutations at position 71. The nature of residue at position 71 influences two main factors responsible for modulating the redox potential; (a) the rotational isomer preference with respect to heme binding and (b) the in-plane rotation of the heme within the heme binding pocket. Thus, following the mutation at position 71 in mammals, it is possible that the two isoforms of CYB5 localized to different organelles and interacted with a new set of binding partners, as determined by their redox potentials, in order to accomplish a wider range of biochemical functions.
dc.format.extent114 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsThis item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
dc.subjectBiochemistry
dc.subjectBiophysics
dc.subjectCytochrome b5
dc.subjectHeme
dc.subjectMolecular dynamics
dc.subjectRedox potential
dc.titleEvolutionary divergence in proteins: experimental and theoretical studies on the stability and electrochemical properties of cytochrome b5
dc.typeDissertation
dc.contributor.cmtememberBenson, David R
dc.contributor.cmtememberRichter, Mark
dc.contributor.cmtememberLamb, Audrey
dc.contributor.cmtememberZhu, Hao
dc.thesis.degreeDisciplineBiochemistry & Molecular Biology
dc.thesis.degreeLevelPh.D.
kusw.oastatusna
kusw.oapolicyThis item does not meet KU Open Access policy criteria.
dc.rights.accessrightsopenAccess


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