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Conversion of a Mechanosensitive Channel Protein from a Membrane-Embedded to a Water-Soluble form by Covalent Modification with Amphiphiles

Becker, Christian F. W. and Strop, Pavel and Bass, Randal B. and Hansen, Kirk C. and Locher, Kaspar P. and Ren, Gang and Yeager, Mark and Rees, Douglas C. and Kochendoerfer, Gerd G. (2004) Conversion of a Mechanosensitive Channel Protein from a Membrane-Embedded to a Water-Soluble form by Covalent Modification with Amphiphiles. Journal of Molecular Biology, 343 (3). pp. 747-758. ISSN 0022-2836. doi:10.1016/j.jmb.2004.08.062.

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Covalent modification of integral membrane proteins with amphiphiles may provide a general approach to the conversion of membrane proteins into water-soluble forms for biophysical and high-resolution structural studies. To test this approach, we mutated four surface residues of the pentameric Mycobacterium tuberculosis mechanosensitive channel of large conductance (MscL) to cysteine residues as anchors for amphiphile attachment. A series of modified ion channels with four amphiphile groups attached per channel subunit was prepared. One construct showed the highest water solubility to a concentration of up to 4 mg/ml in the absence of detergent. This analog also formed native-like, α-helical homo-pentamers in the absence of detergent as judged by circular dichroism spectroscopy, size-exclusion chromatography and various light-scattering techniques. Proteins with longer, or shorter polymers attached, or proteins modified exclusively with polar cysteine-reactive small molecules, exhibited reduced to no solubility and higher-order aggregation. Electron microscopy revealed a homogeneous population of particles consistent with a pentameric channel. Solubilization of membrane proteins by covalent attachment of amphiphiles results in homogeneous particles that may prove useful for crystallization, solution NMR spectroscopy, and electron microscopy.

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Rees, Douglas C.0000-0003-4073-1185
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Additional Information:© 2004 Elsevier. (Received 22 July 2004; received in revised form 19 August 2004; accepted 19 August 2004) We thank R. Spencer (Merck Research Laboratories) for helpful discussions and for the wild-type Tb_MscL gene used in this study, John Philo (Alliance Protein Laboratories) for dynamic light-scattering studies and analysis, and Chris Garcia (Stanford University) and Stephen Mayo (California Institute of Technology) for helpful discussions and suggestions. This work was supported by NIH PO1 GM062532 (to C.B., P.S., D.R., G.K.) and NIH RO1 HL48908 (to M.Y.). During this work G.R. was supported by a California Universitywide AIDS Research Program Fellowship, and M.Y. was supported, in part, by a Clinical Scientist Award in Translational Research from the Burroughs Wellcome Fund.
Funding AgencyGrant Number
NIHPO1 GM062532
NIHRO1 HL48908
California Universitywide AIDS Research Program FellowshipUNSPECIFIED
Burroughs Wellcome FundUNSPECIFIED
Subject Keywords:mechanosensitive channel of large conductance (MscL); water-soluble membrane protein; amphiphiles; light-scattering; electron microscopy
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Record Number:CaltechAUTHORS:20141212-144652104
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ID Code:52782
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Deposited On:22 Dec 2014 21:13
Last Modified:10 Nov 2021 19:43

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