Pressure-Induced Spectral Shifts in GFP Mutants Explained by Molecular Dynamics Simulations

By combining spectroscopic measurements under high pressure with molecular dynamics simulations we investigate how sub-angstrom structural perturbations are able to tune protein function. We monitored the variations in fluorescence output of two Green Fluorescent Protein mutants (termed Mut2 and Mut2Y, the latter containing the key T203Y mutation) subjected to pressures up to 600 MPa, at various temperatures in the 280-320 K range. By performing ~100ns molecular dynamics simulations of the protein structures at various pressures, we evidenced subtle changes in conformation and dynamics around the light-absorbing chromophore. Such changes explain the measured spectral tuning, especially in the case of the sizable 120 cm-1 red-shift observed for pressurized Mut2Y, but absent in Mut2. Previous work by Barstow et al [1] on pressure effects on GFP also involved a T203Y mutant. Using cryocooling X-ray crystallography at high pressure they linked the observed, pressure-induced, fluorescence blue shift at low temperature (77 K) to key changes in relative conformation of the chromophore and Y203 phenol ring. At room temperature, however, they observe a red shift at high pressure, analogous to the one in Mut2Y. Our investigation of structural variations in compressed Mut2Y also explains their result, bridging the gap between low-temperature and room-temperature high-pressure effects.