ArticleBiophysical journal2026
A multiscale screening platform for rapid GPCR variant profiling reveals color-tuned JSR1 mutants.
Article in Biophysical journal, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Integral membrane proteins such as G-protein-coupled receptors (GPCRs), including visual pigments, are critical targets in both fundamental and pharmaceutical research. However, low expression levels and intrinsic instability often hinder their functional and structural characterization. Therefore, efficient screening methods are needed to identify suitable candidates for biochemical and biophysical analysis. Although the insect cell baculovirus expression system is commonly used for the production of functional GPCRs, the virus generation steps are labor intensive, hampering efficient high-throughput screening. In this study, we addressed this limitation by developing a platform for rapid high-titer baculovirus generation in 96-well suspension cultures and subsequent protein expression screening using the same format. The strength of our platform lies in its modular, multiscale design. Beyond the 96-well micro-scale screening phase, we provide protocols for expression of selected variants in 25-mL cultures, followed by a mini-scale affinity purification. The protein yields are sufficient for functional characterization of multiple GPCR variants in parallel using diverse in vitro assays. This intermediate screening step enables data-driven selection of promising candidates for large-scale expression and further characterization. We demonstrate the platform's capabilities through a mutant screen of the light-sensitive GPCR Jumping Spider Rhodopsin 1 (JSR1). From 56 JSR1 mutants, we selected 30 variants for further characterization after expression screening. For all these, we obtained high-quality UV/Vis absorption spectra in both inactive and active states. Remarkably, we discovered five previously uncharacterized mutants with well-separated absorption maxima that are promising candidates for optogenetic applications to switch GPCR signaling on and off with light. Additionally, we show the platform's utility for general functional GPCR assays, including assessment of G-protein activation and thermostability. We anticipate that this workflow will be a valuable resource for the GPCR research community, accelerating selection and characterization of variants with greater efficiency and reliability.
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