Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli.

Crystallography has advanced our understanding of G protein-coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.

Directed evolution of G-protein-coupled receptors for high functional expression and detergent stability.

G-protein-coupled receptors (GPCRs) are cell-surface receptors exhibiting a key role in cellular signal transduction processes, thus making them pharmacologically highly relevant target proteins. However, the molecular mechanisms driving receptor activation by ligand binding and signal transduction are poorly understood, since as integral membrane proteins, most GPCRs are very challenging for functional and structural studies. The biophysical properties of natural GPCRs, usually required by the cell in only low amounts, support their functionality in the lipid bilayer but are insufficient for high-level recombinant overexpression and stability in detergent solution.

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.

Critical features for biosynthesis, stability, and functionality of a G protein-coupled receptor uncovered by all-versus-all mutations.

The structural features determining efficient biosynthesis, stability in the membrane and, after solubilization, in detergents are not well understood for integral membrane proteins such as G protein-coupled receptors (GPCRs). Starting from the rat neurotensin receptor 1, a class A GPCR, we generated a separate library comprising all 64 codons for each amino acid position. By combining a previously developed FACS-based selection system for functional expression [Sarkar C, et al.