Single-Molecule Fluorescence-Based Measurements of Conformational Dynamics of Calcium-Binding Protein Recoverin

Abstract

Recoverin is a calcium-binding protein expressed primarily in the photoreceptor cells of the vertebrate retina, where it participates in the regulation of phototransduction and adaptation of the visual system to background light. Binding of calcium ions to recoverin induces large conformational changes, including the release of its post-translationally attached myristoyl group from a hydrophobic cavity of the protein into the solution. Previously, it was shown that the fluorophore Alexa647 site-specifically bound to a unique cysteine-39 of recoverin can be employed as a reporter of its Ca2+-dependent conformational transitions since these transitions affect fluorescence lifetime, and spectra of the dye. Here, we addressed a question, whether the Ca2+-dependent conformers of recoverin, previously resolved by NMR, can be recorded using single-molecule fluorescence microscopy. To this end, the samples of recombinant bovine recoverin site-specifically labeled at cysteine-39 with either Alexa647 or sulfo-Cy5 maleimide-functionalized fluorophores were prepared. Both forms of the protein exhibited Ca2+-induced conformational changes as revealed by steady-state spectrometry of intrinsic tryptophans fluorescence. Interestingly, two chemically similar cyanine dyes (Alexa647 and sulfo-Cy5) prodruced quite different effects on recoverin state and exhibited different photophysical responses to changes in its conformation. We performed single-molecule experiments with recoverin fluorescently labeled with Alexa647. Using a confocal setup with multiparameter fluorescence detection (MFD) we measured fluorescence lifetimes and anisotropies of bursts from individual recoverin molecules. Observation of individual molecules in solution is limited by their diffusion time (typically, 1-10 ms) - to track slower conformational changes we specifically immobilized recoverin using a biotin-streptavidin tether. Using a TIRF microscope we measured intensity traces from individual immobilized molecules. Our results show that conformational dynamics of recoverin can be measured at the single-molecule level using the protein site-specifically labeled at cysteine-39 with Alexa647. This work is supported by RFBR (№20-34-70034).