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Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

Clayton, Daniel and Shapovalov, George and Maurer, Joshua A. and Dougherty, Dennis A. and Lester, Henry A. and Kochendoerfer, Gerd G. (2004) Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America, 101 (14). pp. 4764-4769. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:CLApnas04

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Abstract

Total chemical protein synthesis was used to generate multimilligram quantities of the mechanosensitive channel of large conductance from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (Tb-MscL). Cysteine residues introduced to allow chemical ligation were masked with cysteine-reactive molecules, resulting in side chain functional groups similar to those of the wild-type protein. Synthetic channel proteins were transferred to 2,2,2-trifluoroethanol and reconstituted into vesicle membranes. Fluorescent imaging of vesicles showed that channel proteins were membrane-localized. Single-channel recordings showed that reconstituted synthetic Ec-MscL has conductance, pressure dependence, and substate distribution similar to those of the recombinant channel. Reconstituted synthetic Tb-MscL also displayed conductance and pressure dependence similar to that of the recombinant protein. Possibilities for the incorporation of unnatural amino acids and biophysical probes, and applications of such synthetic ion channel analogs, are discussed.


Item Type:Article
Additional Information:Copyright © 2004 by the National Academy of Sciences. Edited by William F. DeGrado, University of Pennsylvania School of Medicine, Philadelphia, PA, and approved January 14, 2004 (received for review September 5, 2003). WT recombinant Ec-MscL was provided by Jun-yong Choe and Doug Rees (California Institute of Technology). We thank Erik Rodriguez (California Institute of Technology) for help with studies on the vesicle reconstitution of synthetic Ec-MscL and Christian Becker (Gryphon Therapeutics) for support and discussions. This work was supported by National Institutes of Health Grant GM-062532. This paper was submitted directly (Track II) to the PNAS office.
Subject Keywords:ION-CHANNEL, POTASSIUM CHANNEL, CRYSTAL-STRUCTURE, TERMINAL DOMAIN, PROTEIN, MSCL, RECONSTITUTION, SEMISYNTHESIS, FLUORESCENCE, SELECTIVITY
Record Number:CaltechAUTHORS:CLApnas04
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:CLApnas04
Alternative URL:http://dx.doi.org/10.1073/pnas.0305693101
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:896
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:03 Nov 2005
Last Modified:14 Nov 2014 19:18

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