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A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies
Francesca Magnani1, Maria J Serrano-Vega1, Yoko Shibata1
1MRC Laboratory of Molecular Biology, Cambridge, UK.
Nature Protocols
|July 29, 2016
Summary
We developed a method to stabilize integral membrane proteins (MPs) for structural studies. This approach enhances protein crystallization, enabling detailed analysis and drug design for important targets like G-protein-coupled receptors (GPCRs).
Area of Science:
- Structural Biology
- Biophysics
- Drug Discovery
Background:
- Integral membrane proteins (MPs) are crucial for cellular functions but are often unstable in detergent solutions, hindering structural determination.
- The instability of mammalian MPs poses a significant challenge for crystallization and subsequent structure-based drug design.
- G-protein-coupled receptors (GPCRs) are a major class of MPs targeted for therapeutic interventions but are notoriously difficult to crystallize.
Purpose of the Study:
- To develop a robust strategy for thermostabilizing integral membrane proteins (MPs) to facilitate their crystallization and structural analysis.
- To establish a systematic mutagenesis and assay approach applicable to various MPs, including receptors, ion channels, and transporters.
- To enable structure-based drug design by producing stable MP constructs suitable for determining high-resolution structures.
Main Methods:
- A thermostabilization strategy involving systematic Ala/Leu scanning mutagenesis (generating ~300 mutants).
- Expression of mutants in mammalian cells via transient transfection.
- Thermostability assay using (125)I-labeled radioligand binding to unpurified, detergent-solubilized MPs to identify stable variants.
- Combining individual stabilizing point mutations to create optimally stable MPs.
Main Results:
- Successfully developed a method to enhance the thermostability of integral membrane proteins (MPs).
- Thermostabilized MPs exhibit improved crystallizability, leading to high-quality structural data.
- The methodology is efficient, with G-protein-coupled receptors (GPCRs) taking approximately 6-12 months for stabilization.
Conclusions:
- The developed thermostabilization strategy significantly improves the prospects for crystallizing and determining the structures of challenging integral membrane proteins (MPs).
- This approach facilitates structure-based drug design, particularly for G-protein-coupled receptors (GPCRs), by enabling the study of receptors bound to various ligands.
- The described protocols provide a framework for developing thermostability assays and optimizing MP mutants for structural biology and biophysical studies.
