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Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL) - New gain of function mutations in the loop region

Maurer, Joshua A. and Elmore, Donald E. and Lester, Henry A. and Dougherty, Dennis A. (2000) Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL) - New gain of function mutations in the loop region. Journal of Biological Chemistry, 275 (29). pp. 22238-22244. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:MAUjbc00

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Abstract

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence. Not all of the resulting M. tuberculosis mutants displayed a gain of function phenotype; for instance, normal phenotypes were noted for mutations at Ala20, the analogue of the highly sensitive Gly22 site in E. coli. A previously unnoticed intersubunit hydrogen bond in the extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking studies indicated that these sites are spatially close under physiological conditions. In general, mutation at these positions results in a gain of function phenotype, which provides strong evidence for the importance of the loop region in MscL channel function. No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment. Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL.


Item Type:Article
Additional Information:Copyright © 2000 by the American Society for Biochemistry and Molecular Biology. Received for publication, April 11, 2000, and in revised form, May 5, 2000. http://dx.doi.org/10.1109/50.933286 We are grateful to Prof. Douglas Rees, Dr. Randal Bass, Dr. Geoffrey Chang, Dr. Robert Spencer, and George Shapovalov for helpful discussions. Plasmids and wild type Tb-MscL proteins used in these studies were obtained from the Rees group. MALDI-TOF mass analyses were carried out at the Caltech Protein Microanalytical Laboratory under the direction of Gary M. Hathaway.
Subject Keywords:LARGE-CONDUCTANCE; SINGLE RESIDUE; ION-CHANNEL; PROTEIN; ALIGNMENT
Record Number:CaltechAUTHORS:MAUjbc00
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:MAUjbc00
Alternative URL:http://dx.doi.org/10.1074/jbc.M003056200
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5103
Collection:CaltechAUTHORS
Deposited By: Lindsay Cleary
Deposited On:29 Sep 2006
Last Modified:26 Dec 2012 09:03

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