Mutant GPCR Conformational Stability in Detergents
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Solution Overview
Problem
G protein-coupled receptors (GPCRs) are challenging to crystallize due to their instability in detergent solutions and existence in multiple conformations, which hinders structure determination and drug development.
Innovation Solution
Identifying and introducing specific mutations that enhance the stability and conformational stability of GPCRs, allowing them to maintain a biologically relevant conformation, thereby improving their stability under denaturing conditions and increasing the likelihood of obtaining diffraction-quality crystals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If GPCRs are purified from natural sources, then they maintain native conformation and function, but they exhibit poor stability in detergent solutions leading to immediate denaturation or precipitation
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at defined positions within the GPCR transmembrane domains. These mutations alter the physical-chemical parameters of the protein (hydrophobicity, charge distribution, steric constraints) to enhance stability in detergent environments while preserving the biologically active conformation. The mutations are strategically placed to optimize detergent compatibility without disrupting functional conformations.
Solution Approach 2:
The patent implements local quality by making targeted, position-specific mutations rather than global changes. Specific amino acid residues at critical transmembrane positions are modified to improve detergent stability, while the rest of the protein structure remains unchanged to maintain native conformation and function. This localized approach allows optimization of detergent compatibility without compromising overall protein integrity.
2Ease of manufacture
If detergents with short aliphatic chains and small or charged head groups are used for crystallisation, then crystal growth is promoted, but GPCR stability is severely restricted leading to denaturation or precipitation
Solution Approach 1:
The patent resolves this contradiction by changing the protein's parameters through mutagenesis. The mutated GPCRs have altered surface properties and hydrophobicity patterns that enable them to remain stable in crystallisation-optimized detergents (with short chains and small/charged head groups) that would otherwise denature wild-type proteins. This allows exploration of crystallisation conditions previously inaccessible due to protein instability.
Solution Approach 2:
The patent applies preliminary action by pre-stabilizing the GPCR through mutagenesis before the crystallisation process. The mutations are introduced in advance to confer detergent stability, allowing the protein to withstand the harsh conditions required for crystal growth. This preparatory stabilization enables subsequent crystallisation attempts that would fail with unstable wild-type proteins.
3Productivity
If eukaryotic membrane proteins are overexpressed using standard techniques, then protein yield is improved, but they exhibit poor stability in detergent solutions restricting crystallisation conditions
Solution Approach 1:
The patent applies parameter changes by mutating the eukaryotic membrane protein to alter its physical-chemical properties. The mutations enhance detergent compatibility and conformational stability, allowing the overexpressed protein to maintain stability in detergent solutions. This enables the combination of high productivity from overexpression with the detergent stability required for crystallisation.
Data Source
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AI summary
A method of producing a mutant GPCR with increased conformational stability under denaturing conditions such as heat, a detergent, a chaotropic agent or an extreme of pH, relative to its parent GPCR, the method comprising: (a) identifying in the amino acid sequence of one or more mutants of a first parent GPCR with increased conformational stability under denaturing conditions such as heat, a detergent, a chaotropic agent or an extreme of pH, relative to the first parent GPCR, the position or positions at which the one or more mutants have at least one different amino acid residue compared to the first parent GPCR, and (b) making one or more mutations in the amino acid sequence that defines a second GPCR within a window or windows of i plus or minus 5 residues where i is the corresponding position or positions, to provide one or more mutants of a second parent GPCR with increased conformational stability relative to the second parent GPCR; wherein the second GPCR is able to be aligned with the first GPCR and the corresponding position or positions are as identified by alignment.