Mutant GPCR Stabilization via Transmembrane Helix 7 Mutation
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Solution Overview
Problem
Current methods face challenges in stabilizing eukaryotic membrane proteins, particularly G-protein coupled receptors (GPCRs), which are crucial for drug development but often unstable in detergent solutions, limiting their crystallization and structural determination.
Innovation Solution
Mutating specific amino acid residues within a window of i plus or minus 5 residues, particularly around position 2.46, and in transmembrane helix 7, to increase the stability of GPCRs, making them more amenable to crystallization and drug discovery studies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If GPCRs are used in detergent solutions for structural determination, then the proteins can be purified and crystallized, but the proteins lose stability and denature
Solution Approach 1:
The patent applies parameter changes by systematically mutating amino acid residues at specific positions (particularly position 2.46 and surrounding residues in the transmembrane domain) to alter the protein's physical-chemical properties. These mutations change the stability parameters of the GPCR, enabling it to maintain its native conformation in detergent solutions that would otherwise cause denaturation. The method involves screening mutants with improved thermal stability and detergent resistance while preserving functional activity.
2Stability of the object's composition
If mutations are introduced to increase GPCR stability, then thermal and conformational stability improve, but the risk of losing functional activity increases
Solution Approach 1:
The patent applies local quality by targeting specific local regions of the GPCR protein structure for mutation, rather than random mutagenesis throughout the entire protein. The focus is on the transmembrane domain, particularly position 2.46 and surrounding residues, which are critical for maintaining the structural framework. By localizing mutations to these specific regions, the patent achieves stability improvement while minimizing disruption to other functional domains of the protein.
Solution Approach 2:
The patent employs feedback mechanisms by systematically screening mutant GPCRs for both stability improvement and functional activity retention. The screening process provides feedback on which mutations achieve the desired stability enhancement without compromising function, allowing iterative optimization. Functional assays and stability measurements are used to select mutants that satisfy both criteria, creating a feedback loop that guides the mutagenesis strategy.
Data Source
AI summary
A method for producing a mutant G-protein coupled receptor (GPCR) with increased stability relative to a parent GPCR, the method comprising making one or more mutations in the amino acid sequence that defines a parent GPCR, wherein (i) the one or more mutations are located within a window of/plus or minus 5 residues, where/is the position of amino acid residue 2.46 in the parent GPCR when the parent GPCR is a Class 1 GPCR, or where/is the position of an equivalent amino acid residue in the parent GPCR when the parent GPCR is a Class 2 or 3 GPCR, and/or (ii) the one or more mutations are located within an amino acid sequence of transmembrane helix 7 in the parent GPCR which amino acid sequence interacts with the window of/plus or minus 5 residues, to provide one or more mutants of the parent GPCR with increased stability.