A multiscale screening platform for rapid GPCR variant profiling reveals color-tuned JSR1 mutants

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Integral membrane proteins, including G-protein-coupled receptors (GPCRs), are crucial research targets but often exhibit low expression and instability.
• Current methods for producing functional GPCRs, such as the baculovirus expression system, are labor-intensive, limiting high-throughput screening.

Purpose of the Study:

• To develop an efficient platform for rapid screening and characterization of G-protein-coupled receptor (GPCR) variants.
• To overcome limitations in high-throughput screening of GPCRs for biochemical and biophysical analysis.

Main Methods:

• A modular, multiscale platform was developed for rapid high-titer baculovirus generation in 96-well suspension cultures.
• The platform supports protein expression screening, intermediate-scale expression (25-mL cultures), and mini-scale affinity purification.
• Functional assays, including UV/Vis spectroscopy, G-protein activation, and thermostability, were employed.

Main Results:

• The platform successfully screened 56 mutants of Jumping Spider Rhodopsin 1 (JSR1), selecting 30 variants for further analysis.
• High-quality UV/Vis absorption spectra were obtained for selected JSR1 variants in both inactive and active states.
• Five novel JSR1 mutants with distinct absorption maxima were identified as promising candidates for optogenetic applications.

Conclusions:

• The developed platform significantly accelerates the selection and characterization of functional GPCR variants.
• This workflow provides a valuable resource for GPCR research, enhancing efficiency and reliability in identifying candidates for drug discovery and fundamental studies.
• The platform facilitates the discovery of novel GPCR variants with potential applications in optogenetics and other functional assays.