G-Protein-Coupled Receptors (GPCR) are membrane protein receptors involved in most eukaryotic signaling processes via interactions with hormones, neurotransmitters, cytokines, etc. All GPCR have similar structures composed of seven membrane-spanning domains, three extracellular and intracellular loops, and extracellular N-terminal and intracellular C-terminal domains. GPCR comprise the largest pharmaceutically relevant membrane protein group, serving as targets for approximately one-third of all drugs currently approved by the FDA and approximately one-fifth of those presently undergoing clinical trials. Still, only about one-fourth of all known GPCR are current pharmaceutical targets, leaving vast untapped potential. Studying membrane proteins is complicated by their hydrophobicity, instability when removed from membranes, and difficulties with expression, purification, and crystallization. Recent studies show proof of principle for water-soluble GPCR analogs by substituting hydrophobic amino acid residues with residues expected to have similar secondary structure in a soluble protein of comparable size. The published example utilized a target-specific design, whereas, in this study, class-wide, as well as receptor-specific strategies are being developed for application to larger numbers of class A GPCR. The first strategy relies on an informatic analysis of hydrophobic surface patches in 50 class A GPCR to identify common hydrophobic surface sites. In the receptor-specific approach, tailored sets of hydrophobic surface sites were identified. A QTYS substitution code (L to Q; I, V to T; F to Y; A to S) was applied to disrupt these hydrophobic surface patches with the intent to alter environmental preference without altering overall tertiary structure. Computational application of up to 42 or 65 substitutions at common hydrophobic sites lowered hydrophobic surface area of representative class A GPCR analyzed by an average of 34% and 49%, respectively. Experimental assessment of substitution strategies is in progress using the dopamine receptor 2 (D2), adenosine diphosphate receptor (P2Y12), and cannabinoid receptor 1 (CB1) as test cases. GPCR analog fusions with an emerald green fluorescent protein (EmGFP) reporter, optimized for <i>E. coli</i> codon usage, are being used in bacterial expression trials.  EmGFP only fluoresces if the upstream fusion partner is correctly folded, providing a rapid and efficient screening strategy for protein expression.

Water-soluble G-protein coupled receptor analogs: Systematic disruption of hydrophobic surface patches to optimize expression

ACS Spring 2022

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