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PEDS Advance Access published online on March 23, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi010
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received November 19, 2004
Revised February 14, 2005
Accepted February 15, 2005

Article

Redesigning an integral membrane K+ channel into a soluble protein

Tarmo P. Roosild 1 and Senyon Choe 1*

1 Structural Biology Laboratory, Salk Institute and Division of Biology, University of California at San Diego, San Diego, CA 92037, USA

* To whom correspondence should be addressed.
Senyon Choe, E-mail: choe{at}salk.edu


  Abstract

Even though the structure determination of soluble proteins has become routine, the number of unrelated integral membrane protein structures remains at a few dozen. The importance of this class of proteins to the molecular mechanisms underlying numerous biological phenomena demands that novel experimental techniques be developed to overcome the limitations imposed by conventional detergent-dependent approaches. Here we report the re-engineering of a putative K+ channel protein of unknown structure into a water-soluble analogue. By analyzing evolutionary conservation patterns of related sequences, lipid-facing residues of the primitive channel were identified and mutagenized into more polar alternatives. Further stabilization of the resultant construct was achieved through fusion with maltose-binding protein. The final soluble protein forms a tetramer, suggesting that it accurately models its predecessor. This methodology, as a viable alternative to the use of detergents, should be applicable to a wide range of integral membrane protein families including transporters and other signal transducers.

Keywords: integral membrane protein; maltose binding protein-membrane protein crystallization; potassium channel.
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