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Determination of transmembrane protein structure by disulfide cross-linking: The Escherichia coli Tar receptor

Pakula, Andrew A. and Simon, Melvin I. (1992) Determination of transmembrane protein structure by disulfide cross-linking: The Escherichia coli Tar receptor. Proceedings of the National Academy of Sciences of the United States of America, 89 (9). pp. 4144-4148. ISSN 0027-8424. PMCID PMC525649. doi:10.1073/pnas.89.9.4144.

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We have devised a generally applicable strategy for analysis of protein structure and have applied it to examine the structure of the transmembrane portion of the Tar receptor of Escherichia coli. The basis of our approach is the use of disulfide cross-linking to identify residues that are within close proximity. To generate and test large numbers of cysteine pairs, we used an unusual method of mutagenesis by which cysteine substitutions can be created randomly at a number of targeted codons. Cysteine-substituted proteins encoded by mutagenized genes may be screened directly for disulfide formation within oligomers or, alternatively, different pools of genes may be randomly recombined to generate gene populations with substitutions in multiple regions. Thus, it is possible to detect a variety of disulfide cross-links between and within individual protein molecules. Interactions between the four membrane-spanning stretches of the Tar dimer were probed by measuring the tendency of 48 cysteine substitutions throughout this region to form disulfide cross-links with one another. We have interpreted these data to suggest a helical-bundle structure for the transmembrane region. The four helices of this bundle are not structurally equivalent: the two TM1 helices interact closely, whereas the TM2 helices are more peripherally located.

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Additional Information:© 1992 by the National Academy of Sciences. Contributed by Melvin I. Simon, January 30, 1992. We thank David Botstein for communication of unpublished methods for oligonucleotide synthesis; James Bowie and Yun Sun for suggestions on molecular modeling calculations; AnnaMarie Aquinaldo, Conrad Sevilla III, and Brett Znider for oligonucleotide synthesis; Robert Bourret, Doug Rees, and Andy Myers for helpful comments and discussion. This work was supported by National Institutes of Health Grant AI 19296 and by a grant from the Markey Foundation. A.A.P. is the recipient of a postdoctoral fellowship from the American Cancer Society, California division. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Funding AgencyGrant Number
NIHAI 19296
Lucille P. Markey Charitable TrustUNSPECIFIED
American Cancer Society, California DivisionUNSPECIFIED
Subject Keywords:chemotaxis; signal transduction; helical bundle
Issue or Number:9
PubMed Central ID:PMC525649
Record Number:CaltechAUTHORS:PAKpnas92
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9212
Deposited By: Tony Diaz
Deposited On:27 Nov 2007
Last Modified:08 Nov 2021 20:57

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