Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution.
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® 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.
Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
Karola M
KM
Schlinkmann
Matthias
M
Hillenbrand
Alexander
A
Rittner
Madeleine
M
Künz
Ralf
R
Strohner
Andreas
A
Plückthun
D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003902:Detergents / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008823:Micelles; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018028:Receptors, Neurotensin / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008823:Micelles; D043562:Receptors, G-Protein-Coupled; D018028:Receptors, Neurotensin; C043426:inner membrane protein, E coli; C114036:neurotensin type 1 receptor; D019204:GTP-Binding Proteins