Protein function depends on structure and oligomerization, yet dynamic conformational changes in membrane proteins such as G protein–coupled receptors remain difficult to study using traditional methods. This work develops novel site-directed fluorescence labeling approaches that exploit distance-dependent bimane quenching by tryptophan residues to probe protein secondary structure, tertiary constraints, and real-time conformational changes. A thiol-cleavable fluorophore, PDT-bimane, enables automated, high-throughput structural analysis. These methods are applied to visual rhodopsin, revealing a strong preference for higher-order oligomerization in membrane environments.
