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Updated: Jun 30, 2026
09:12
G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay
Published on: September 10, 2016
Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity
Casim A Sarkar1, Igor Dodevski, Manca Kenig
1Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Summary
We developed a directed evolution method to improve integral membrane protein expression and stability. This technique enhanced a G protein-coupled receptor
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Integral membrane proteins, such as G protein-coupled receptors (GPCRs), are crucial drug targets but challenging to study due to difficulties in expression and purification.
- Directed evolution offers a powerful approach to engineer proteins with improved properties.
Purpose of the Study:
- To develop and apply a novel directed evolution method for enhancing the functional expression and stability of integral membrane proteins.
- To engineer a mammalian GPCR for improved heterologous expression and biophysical characteristics.
Main Methods:
- Utilized directed evolution in Escherichia coli to generate variants of a mammalian G protein-coupled receptor.
- Screened for improved functional expression, stability, and ligand-binding properties.
- Assessed heterologous expression in eukaryotic systems (Pichia pastoris, HEK293T cells).
Main Results:
- Achieved an order-of-magnitude increase in functional expression for the target GPCR while maintaining wild-type biochemical properties.
- Demonstrated significantly enhanced heterologous expression in Pichia pastoris (12-fold) and HEK293T cells (3-fold).
- Identified a single amino acid substitution that selectively abolishes antagonist binding while preserving agonist binding.
Conclusions:
- The developed directed evolution method is effective for improving integral membrane protein expression, stability, and function.
- Engineered GPCR variants show promise for structural studies by overcoming current bottlenecks in protein production and characterization.
- This approach facilitates the generation of stable protein variants in specific conformational states.
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