Engineering an ultra-thermostable β(1)-adrenoceptor

Area of Science:

• Biochemistry
• Structural Biology
• Pharmacology

Background:

• Conformational thermostabilisation is key for determining G-protein-coupled receptor (GPCR) structures.
• Thermostable GPCR mutants facilitate crystallography by tolerating detergents and adopting single conformations.
• The β(1)-adrenoceptor mutant (β(1)AR-m23) was the first thermostabilised receptor structure determined.

Purpose of the Study:

• To further enhance the thermostability of the β(1)AR-m23 mutant.
• To investigate the impact of additional mutations on receptor stability and properties.
• To identify optimal strategies for GPCR thermostabilisation.

Main Methods:

• Introduction of three specific mutations (I129V, D322K, Y343L) into β(1)AR-m23.
• Evaluation of thermostability using differential scanning fluorimetry in various detergents.
• Assessment of pharmacological properties and stability in short-chain and denaturing detergents.

Main Results:

• A triple mutant of β(1)AR-m23 exhibited a 31°C increase in thermostability compared to wild-type and a 13°C increase over the original mutant.
• Engineered salt bridges and leucine scanning mutagenesis proved most effective for improving thermostability.
• The enhanced mutant maintained pharmacological properties and showed increased stability in detergents like heptylthioglucoside and SDS.

Conclusions:

• Additional mutations can significantly enhance GPCR thermostability beyond initial stabilisation efforts.
• Thermostable GPCR mutants are crucial tools for structural biology, enabling studies in various detergent conditions.
• Optimised thermostabilisation strategies are vital for advancing GPCR structure determination and drug discovery.