Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 422, Issue 3, Pages 414-428Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2012.05.039
Keywords
in vitro DNA recombination; screening and selection; plasmid copy number; membrane protein; expression; coevolution
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Funding
- NCCR Structural Biology (Swiss National Science Foundation)
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To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several shift positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics (R) technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution. (c) 2012 Elsevier Ltd. All rights reserved.
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