An optimized purification and reconstitution method for the MscS channel: strategies for spectroscopical analysis

Vasquez, V., Cortes, D. M., Furukawa, H., Perozo, E. (2007) An optimized purification and reconstitution method for the MscS channel: strategies for spectroscopical analysis. Biochemistry, 46 (23). pp. 6766-73. ISSN 0006-2960 (Print)0006-2960 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/17500538
DOI: 10.1021/bi700322k

Abstract

The mechanosensitive channel of small conductance (MscS) plays a critical role in the osmoregulation of prokaryotic cells. The crystal structure of MscS revealed a homoheptamer with three transmembrane segments and a large cytoplasmic domain. It has been suggested that the crystal structure depicts an open state, but its actual functional conformation remains controversial. In the pursuit of spectroscopical approaches to MscS gating, we determined that standard purification methods yield two forms of MscS, with a considerable amount of unfolded channel. Here, we present an improved high-yield purification method based on Escherichia coli expression and a biochemical characterization of the reconstituted channel, optimized to yield approximately 4 mg of a single monodisperse product. Upon reconstitution into lipid vesicles, MscS is unusually prone to lateral aggregation depending on the lipid composition, particularly after sample freezing. Strategies for minimizing MscS aggregation in two dimensions for spectroscopic analyses of gating have been developed.

Item Type: Paper
Uncontrolled Keywords: Crystallography X-Ray Electron Spin Resonance Spectroscopy Escherichia coli physiology Escherichia coli Proteins chemistry isolation & purification metabolism Ion Channels chemistry isolation & purification metabolism Kinetics Liposomes Models Molecular Protein Conformation Software
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > ion channel
organs, tissues, organelles, cell types and functions > tissues types and functions > membranes
biotechnology > chromatography > protein purification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein structure rendering
Investigative techniques and equipment > x ray crystallography
CSHL Authors:
Communities: CSHL labs > Furukawa lab
Depositing User: Leigh Johnson
Date Deposited: 26 Mar 2012 17:57
Last Modified: 31 Aug 2017 18:48
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25163

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