Probing the Impact of Evolutionary Divergence on Structure, Function, Stability and Dynamics of Cytochrome b5

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Progress in bioinformatics has revealed a single gene in insects and plants that codes for cytochrome b5 (b5), a membrane-anchored electron transfer heme protein. Duplication of an analogous gene and subsequent evolutionary divergence has provided mammals with two b5 isoforms, each playing specialized roles in distinct sub-cellular organelles. The isoform that resides in the outer mitochondrial membrane (OM b5) is considerably more stable than its counterpart in the endoplasmic reticulum (microsomal, or Mc b5), and also has a more negative reduction potential, a parameter related to function in electron transfer proteins. The lone b5 from house fly (HF b5) exhibits intermediate stability and redox properties. This project will build upon successful efforts over the past several years, aimed at understanding the nature of the divergent biophysical and functional properties of b5 isoforms, with a new emphasis on the critical role played by polypeptide dynamics. Hence, these studies are aimed at probing a hypothesis that dissociation of heme from b5 is governed by high amplitude low-frequency cooperative polypeptide motions, which differ for b5 isoforms from different evolutionary lineages. To this end, an experimental approach based on native-state hydrogen-deuterium exchange monitored by NMR spectroscopy will be combined with an emerging computational methodology (replica exchange molecular dynamics; REX-MD) that greatly accelerates the sampling rate of molecular conformations in comparison to previous methods. The dynamic properties exhibited by the polypeptide in a given b5 isoform are ultimately governed by the nature and strength of its interactions with the heme, necessitating that a comprehensive study encompass both the holo (heme-bound) and apo (heme-free) states of the protein. Consequently, the solution structure of rat OM apo-b5 will be determined by NMR spectroscopy and compared to the published structure of rat Mc apo-b5. Insights gained in this manner will be refined through corresponding REX-MD simulations. The third objective is to test the developing understanding of factors affecting stability and dynamics in b5 through the rational design of rat OM b5 mutants with stability greater than that of the wild-type protein. In addition to providing insight into nature's solutions to tuning the properties of b5 for its roles in different organisms, results from this project hold promise of a broader impact by (1) improving general understanding of the factors that stabilize cofactor-containing proteins from thermophilic organisms relative to those of their mesophilic counterparts; and (2) the related ability to rationally design heme proteins with useful applications, such as electron carriers in bioreactors operating at elevated temperatures. Further, the PIs routinely use recent findings from the project in the classroom, both to illustrate concepts and to highlight the reality of how science is done. This can be an effective means of stimulating undergraduate student interest in undertaking research projects, both in the PIs laboratories and in those of their colleagues. All undergraduates participating in the project are encouraged to discuss their results in house and, when possible, to present their results at regional and national meetings.