Mutant Transmembrane Proteins for Crystallization
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Solution Overview
Problem
Current methods face challenges in obtaining high-quality crystal structures of mammalian membrane proteins due to their poor stability and low expression levels, particularly for transmembrane transporters like the cocaine-sensitive rat serotonin transporter (SERT), which are unstable in detergent solutions, limiting the development of new therapies for CNS disorders.
Innovation Solution
Mutations are introduced at the interfaces between transmembrane alpha-helices or in kinked regions of SERT to enhance conformational stability, allowing for the production of thermostabilized mutants that can withstand detergent solubilization and maintain functional activity, facilitating crystallization and drug discovery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If eukaryotic membrane proteins are expressed in detergent solutions, then they can be purified and crystallized, but their conformational stability is poor and they aggregate
Solution Approach 1:
The patent applies parameter changes by systematically mutating amino acid residues at specific positions (interface regions between transmembrane helices) to alter the protein's conformational stability. This involves changing physical-chemical properties of residues at critical interfaces to enhance stability in detergent solutions while maintaining crystallization capability
Solution Approach 2:
The patent applies local quality by focusing mutations specifically at interface regions between transmembrane helices rather than throughout the entire protein. This localized approach targets the specific areas where stability is needed most, preserving other functional regions of the protein
2Stability of the object's composition
If mutations are introduced throughout the entire GPCR to improve stability, then conformational stability increases, but the mutations are scattered and lack a common motif
Solution Approach 1:
The patent identifies and targets specific local regions (interface regions between transmembrane helices) for mutation, creating a systematic pattern rather than scattered random mutations. This local quality approach focuses stability-enhancing changes at structurally critical interfaces
Solution Approach 2:
The patent segments the GPCR into distinct functional regions and identifies interface regions between transmembrane helices as critical zones for mutation. This segmentation allows systematic targeting of specific segments (interface regions) rather than treating the entire protein uniformly
3Productivity
If bacterial membrane proteins are used instead of eukaryotic proteins, then expression and stability improve, but they lack relevance to human physiology and disease
Solution Approach 1:
The patent applies parameter changes to eukaryotic membrane proteins by introducing specific mutations at interface regions, thereby altering their stability parameters to match or exceed bacterial protein performance while maintaining eukaryotic protein identity and physiological relevance
Solution Approach 2:
The patent successfully copies the stability characteristics of bacterial membrane proteins into eukaryotic membrane proteins through targeted mutations, creating eukaryotic proteins with bacterial-like stability and expression properties while retaining human physiological relevance
Data Source
AI summary
The present invention relates to mutant transmembrane proteins which have increased conformational stability when compared to their parent protein, methods of selection and production. In particular the invention relates to mutant transmembrane proteins which are mutated in or in the proximity of the transmembrane alpha helices or in a kinked region or in an alpha-helix adjacent to a kink. The mutant transmembrane proteins have use in crystallisation studies and also in screening to identify compounds for use in drug discovery and therapy.