Experimental and computational investigations of the stability and dynamics of cytochrome bb5s

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Experimental and computational investigations of the stability and dynamics of cytochrome bb5s

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Title: Experimental and computational investigations of the stability and dynamics of cytochrome bb5s
Author: Storch, Elizabeth Marie
Abstract: Both computational and experimental approaches were used to study cytochrome b$\sb5.$ The goal was to investigate protein dynamics and how they may relate to stability and biological function.Computational studies used molecular dynamics simulations to model the solution structure of cytochrome b$\sb5.$ Simulations revealed conformational changes on the protein surface that resulted in the periodic formation of a large cleft. The cleft was located near acidic residues implicated in electrostatic-based protein-protein recognition and complex formation.Simulations of rat and bovine apocytochrome b$\sb5$ were performed to investigate structural consequences of heme removal and the effects of species sequence differences. The simulated rat apoprotein was in good agreement with experimental data, while the variant residues were responsible for the simulated bovine apoprotein deviations from rat experimental data. In both proteins, core 1 exhibited increased mobility, and loss of secondary structure, while core 2 was well maintained and retained native-like structure.Based on the results from the holocytochrome b$\sb5$ simulations, S18D and S18C/R47C mutant proteins were modeled, constructed using site-directed mutagenesis, and then experimentally tested to determine if mutations spanning the cleft region could affect the predicted dynamics.Thermodynamic stability of the proteins in the presence of increasing urea or temperature was studied using absorbance and fluorescence spectroscopies. The mutations had modest effects on heme binding; however, they had pronounced effects on the conformational dynamics prior to heme release. The two spectroscopies supported a complex, three-state denaturation pathway, in contrast to the currently proposed two-state model.Fluorescence was used to determine mutational effects on protein dynamics. Steady-state measurements showed blue-shifted spectra for the mutants. Acrylamide quenching revealed decreased K$\sb{SV}$ values in the mutants, particularly for S18C/R47C at 50$\sp\circ$C. Bimolecular rate constants were calculated from life-time measurements, and at 50$\sp\circ$C, S18C/R47C had a lower rate constant relative to wild-type.Observations from the computational and experimental studies showed the two techniques to be complementary, and the final results would not have been achieved using one technique over the other. Observed differences between the proteins were consistent with the hypothesis that rationally designed mutations may affect localized dynamics and stability in cytochrome b$\sb5.$
Description: Thesis (Ph. D.)--University of Washington, 1998
URI: http://hdl.handle.net/1773/8155

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