PEDS Advance Access originally published online on March 23, 2005
Protein Engineering Design and Selection 2005 18(2):79-84; doi:10.1093/protein/gzi010
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Redesigning an integral membrane K+ channel into a soluble protein
Structural Biology Laboratory, Salk Institute and Division of Biology, University of California at San Diego, San Diego, CA 92037, USA
1 To whom correspondence should be addressed. E-mail: choe{at}salk.edu
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.
Received November 19, 2004; revised February 14, 2005; accepted February 15, 2005.
Edited by William DeGrado