Identification of subdomains in the F and G helices of bacteriorhodopsin that regulate the conformational transitions of the reprotonation mechanism
Date of Award
Doctor of Philosophy (Ph.D.)
First Committee Member
George J. Turner, Committee Chair
We have developed a high-throughput screening method that enables the analysis of bacteriorhodopsin (BR) in whole cell pastes. Reflectance spectra from small volumes of H. salinarum cell culture show close correspondence to those obtained from purified BR. We demonstrate accurate determination of lambdamax values for ground state, light- and dark-adapted BR. Using cells expressing the BR mutant, D85N, we monitored transitions between the M, N, and O intermediate state homologues as a function of pH. We demonstrate that the phenotypes of three mutants (D85N/T170C, D85N/D96N, and D85N/R82Q) previously characterized for their effect on photocycle transitions are reproduced in the whole cell samples. These studies illustrate the correspondence between the pH-dependent ground state transitions accessed by D85N and the transitions accessed by the wild type protein following photoexcitation. We demonstrate that whole cell reflectance spectroscopy can be used to efficiently characterize the large numbers of mutants generated by engineering strategies that exploit saturation mutagenesis.We have performed cysteine-scanning mutagenesis of the bacteriorhodopsin mutant, D85N, to explore the role of individual amino acids in the conformational transitions of the reprotonation mechanism of the BR photocycle. We have systematically replaced each amino acid in the F and G helices and the intervening loop region with a cysteine residue and used whole cell reflectance spectroscopy to evaluate the spectral properties of these mutants. Cysteine mutants were grouped into one of six phenotypes based on the spectral changes associated with their M ↔ N ↔ O intermediate state transitions. Mutations that produced similar phenotypes were found to cluster in discrete molecular domains indicating that M, N, and O possess distinct structures and that unique molecular interactions regulate the transitions between them. The distribution of these domains suggests that: (1) the extramembranous loop region is involved in the stabilization of the N and M intermediates, (2) lipid-protein interactions play a key role in the accumulation of N, and (3) the amino acid side chain interactions in the extracellular portion of he interface between the G and A helices participate in the accumulation of M.
Martinez, Lynell Christine, "Identification of subdomains in the F and G helices of bacteriorhodopsin that regulate the conformational transitions of the reprotonation mechanism" (2002). Dissertations from ProQuest. 1832.