The Journal of General Physiology
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Published 1 August 2001. doi:10.1085/jgp.118.2.193
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© The Rockefeller University Press, 0022-1295/2001//193/ $5.00
Journal of General Physiology, Volume 118, Number 2, 2001

Original Article

Site-Directed Spin-Labeling Analysis of Reconstituted Mscl in the Closed State

Eduardo Perozoa, Anna Klodab, D. Marien Cortesa, and Boris Martinacb

a Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, 22906
b Department of Pharmacology, Queen Elisabeth II Medical Center, University of Western Australia, Crawley, WA 6009, Australia
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, 22906.(434) 982-1616 E-mail: eperozo@virginia.edu

The mechanosensitive channel from Escherichia coli (Eco-MscL) responds to membrane lateral tension by opening a large, water-filled pore that serves as an osmotic safety valve. In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer. Structural information was derived from an analysis of probe mobility, residue accessibility to O2 or NiEdda and overall intersubunit proximity. For the majority of the residues studied, mobility and accessibility data showed a remarkable agreement with the Mycobacterium tuberculosis crystal structure, clearly identifying residues facing the large water-filled vestibule at the extracellular face of the molecule, the narrowest point along the permeation pathway (residues 21–26 of Eco-MscL), and the lipid-exposed residues in the peripheral transmembrane segments (TM2). Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions. However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.

Key Words: mechanosensitive channel • solvent accessibility • dipolar coupling • EPR • transmembrane • segments

© 2001 The Rockefeller University Press


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