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Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography
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Small-scale approach for precrystallization screening in GPCR X-ray crystallography
Martin Audet1,2, Kelly Villers3, Jeffrey Velasquez3
1Departments of Biological Sciences and Chemistry, Bridge Institute, USC Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, California, USA. martin.audet2@usherbrooke.ca.
Nature Protocols
|December 1, 2019
Summary
Determining G protein-coupled receptor (GPCR) structures is challenging. This study introduces a rapid, small-scale screening method to assess GPCR expression and stability, aiding drug discovery efforts.
Area of Science:
- Structural biology
- Biochemistry
- Pharmacology
Background:
- G protein-coupled receptors (GPCRs) are crucial pharmaceutical targets, but their structural determination is hindered by low expression, purification difficulties, and instability.
- Optimizing GPCR protein sequences and purification protocols is complex and time-consuming, complicating the precrystallization phase.
Purpose of the Study:
- To develop a streamlined, small-scale precrystallization screening procedure for GPCRs.
- To enable efficient assessment of GPCR expression levels and in vitro stability prior to large-scale crystallization trials.
Main Methods:
- Flow cytometry was used to detect GPCR expression in Spodoptera frugiperda (Sf9) cell cultures.
- Size-exclusion chromatography and UV absorbance measurements were employed to evaluate GPCR stability.
- The protocol utilizes minimal Sf9 cell culture volumes for intrinsic UV absorbance analysis.
Main Results:
- A procedure was established for small-scale precrystallization screening of GPCRs.
- The method allows for rapid detection of expression and stability using limited material.
- The protocol is compatible with conditions for subsequent scale-up for crystallization.
Conclusions:
- This 8-day protocol provides a robust method for assessing GPCR expression and stability.
- The technique facilitates medium-throughput screening, accelerating the structural determination of GPCRs.
- This approach simplifies the precrystallization process for GPCRs, aiding pharmaceutical research.
