Monitoring GPCR Conformation with GFP-Inspired Dyes

Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research. In this study, we used synthetic analogues of the green fluorescent protein (GFP) chromophore to investigate conformational changes in proteins. We synthesized thiol-reactive derivatives of four GFP chromophore analogues that exhibit solvatochromism. We used these derivatives to label recoverin and the A2A adenosine receptor (A2AAR). Two of these dyes showed Ca2+-induced fluorescence changes when attached to recoverin. Notably, our best-performing DyeC revealed agonist-induced changes in both intensity and shape of the fluorescence spectrum when attached to A2AAR; none of these effects were observed with cyanine and rhodamine environmentally sensitive dyes. Molecular dynamics simulations provided insights into these changes, showing activation of the receptor led to a more confined and hydrophilic environment for DyeC. Additionally, an allosteric modulator induced DyeC fluorescence changes, indicating a distinct receptor conformation. Our study demonstrates GFP-inspired dyes are effective for detecting structural changes in G protein-coupled receptors (GPCRs), with advantages such as intensity-based and ratiometric tracking, red-shifted fluorescence spectra, and sensitivity to allosteric modulation. Our study unveils previously unexplored capacities of the renowned fluorescent core, expanding the toolbox for tracking ligand-induced changes and facilitating new insights into conformational changes induced by allosteric modulators in GPCRs.

Content:
- input, input structures and topologies;
- equilMD, trajectories of equilibrium molecular dynamics simulations (two replicates for each system) in active/inactive states;
- metaMD, trajectories of metadynamics molecular dynamics simulations (two replicates for each system) in active/inactive states.