Probing the Dynamics of Red Fluorescent Protein Variants using NMR Spectroscopy: How Directed Development Affected Protein Dynamics

Fluorescent proteins are commonly used genetically encodable tools for probing intracellular events in real time. Red fluorescent proteins (RFPs) are particularly useful because scattering and absorption are reduced in cells excited at longer wavelengths. Development of RFPs with increased quantum yield and wavelength of emission, among other photophysical properties, is an active area of research; but the optimization of one property is often accompanied by other deleterious effects. This work explores how differences in the photophysical properties of various RFPs may arise from changes in protein dynamics as a product of protein directed development. NMR relaxation experiments were used to probe ps-ns dynamics for the backbone amides, as well as the chromophore tyrosine CβH of mCherry, mRaspberry and mRojoB, three closely related monomeric RFPs with different quantum yields. Results indicate restricted dynamics in the backbone amides and the chromophore tyrosine CβH of the three monomeric RFPs tested. The results of NMR relaxation dispersion experiments suggested differences in \u26mus-ms timescale dynamics in the backbone amides and δ, δ, and γ methyls of isoleucine, leucine and valine residues, respectively, among the same three RFPs. Hydrogen-deuterium exchange experiments were used to show that tdTomato, a dimeric RFP with a high quantum yield, has a higher propensity for local unfolding compared with monomeric mCherry and mRojoB, which have substantially lower quantum yields suggesting a small degree of local unfolding does not abolish fluorescence quantum yield in FPs. Differences in the temperature dependence of fluorescence and secondary structure were observed using fluorescence and CD spectroscopy supporting the idea that directed development affected global stability. How the directed development of RFPs with optimized photophysical properties affects protein dynamics is discussed