Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution inEscherichia coli

• DOI: 10.1073/pnas.1317903111
• Publication Date: 2014-01-23T03:42:30Z
• Authors: Pascal Egloff; Matthias Hillenbrand; Christoph Klenk; Alexander Batyuk; Philipp Heine; Stefanie Balada; Karola M. Schlink...; Daniel J. Scott; Marco Schütz; Andreas Plückthun
• Keywords: Models, Molecular; 1000 Multidisciplinary; 0303 health sciences; Sequence Homology, Amino Acid; Protein Stability; Molecular Sequence Data; Crystallography, X-Ray; 3. Good health; 03 medical and health sciences; 10019 Department of Biochemistry; Escherichia coli; 570 Life sciences; biology; Receptors, Neurotensin; Amino Acid Sequence; Directed Molecular Evolution; Signal Transduction

SignificanceOnly a tiny fraction (<2%) of the unique structures in the protein database correspond to membrane proteins, and only a few of these are of eukaryotic origin, representing potential drug targets. The difficulties in structure determination of these proteins are due to two specific complications, which are unique for membrane proteins: first, low expression levels and, second, the necessity for detergent micelles, which are often destabilizing as they mimic the hydrophobic membrane environment only poorly. We prove that directed evolution has the potential to overcome these problems by determining several structures of evolved eukaryotic G protein–coupled receptor variants. High functional expression levels and superior receptor stability in harsh detergents allowed us to gain deeper insights into this important receptor family.