Abstract
Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research. Here we used thiol-reactive solvatochromic analogs of the green fluorescent protein (GFP) chromophore to track conformational changes in two proteins, recoverin and the A2A adenosine receptor (A2AAR). Two dyes showed Ca2+-induced fluorescence changes when attached to recoverin. Our best-performing dye, DyeC, exhibited agonist-induced changes in both intensity and shape of its fluorescence spectrum when attached to A2AAR; none of these effects were observed with other common environment-sensitive dyes. Molecular dynamics simulations showed that activation of the A2AAR led to a more confined and hydrophilic environment for DyeC. Additionally, an allosteric modulator of A2AAR induced distinct fluorescence changes in the DyeC spectrum, indicating a unique receptor conformation. Our study demonstrated that GFP-inspired dyes are effective for detecting structural changes in G protein-coupled receptors (GPCRs), offering advantages such as intensity-based and ratiometric tracking, redshifted fluorescence spectra, and sensitivity to allosteric modulation.
Keywords:
Biochemistry; Biophysics; Structural biology.