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Subdomains in the F and G helices of bacteriorhodopsin regulate the conformational transitions of the reprotonation mechanism
Author(s) -
Martinez Lynell C.,
Thurmond Robin L.,
Jones Philip G.,
Turner George J.
Publication year - 2002
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.10158
Subject(s) - bacteriorhodopsin , mutant , mutagenesis , chemistry , biophysics , cysteine , amino acid , stereochemistry , transition (genetics) , crystallography , side chain , biology , biochemistry , enzyme , membrane , gene , organic chemistry , polymer
Abstract 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. We have used whole‐cell reflectance spectroscopy to evaluate the spectral properties of the 59 mutants generated during a scan of the entire F and G helices and the intervening loop region. Cys 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 and indicate 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 the interface between helices G and A participate in the accumulation of M. Proteins 2002;48:269–282. © 2002 Wiley‐Liss, Inc.