Time-resolved protein dynamics using x-ray crystallography and optical spectroscopy

Reversibly switchable fluorescent proteins (RSFPs) switch between a highly and weakly fluorescent state upon photoexcitation. This switching is known to be driven by reversible photoisomerisation and a change in protonation state of the chromophore. However, the exact sequence of events after optical excitation is unclear. In this thesis, time-resolved crystallography and vibrational spectroscopy are used to study the ultrafast dynamics of a new RSFP. Time-resolved serial femtosecond crystallography (TR-SFX) is used to probe the trans/cis reaction and reveals ultrafast sub-Angstrom motions of the chromophore and surrounding hydrogen bonding network. The addition of a stokes pulse (shown to dump the excited state population) increases these displacements and allows assignment of the light-induced signals to a vibrationally excited electronic ground state structure instead of an excited state structure. Temperature analysis of the steady-state switching rates resolves an intermediate ground-state structure using conventional crystallography which supports the TR-SFX assignments. Ultrafast pump-probe vibrational spectroscopy confirms that excited state photoisomerisation occurs (~70 ps), proceeded by rearrangement of a nearby arginine residue (~7 ps) with deprotonation (trans-neutral/cis-anion) occurring between 2 ns - 1 ms. An additional dump pulse is then shown to repress product formation and reforms the ground state, further supporting the structural observations of the pump-dump-probe crystallography measurements. These results improve our understanding of photoswitching in RSFPs and have important implications for structural assignments in future TR-SFX experiments. Time-resolved serial synchrotron crystallography (TR-SSX) is an accessible and emerging technique for studying protein dynamics on millisecond timescales. Here, the trans/cis isomerisation of an RSFP is probed at 10 ms with a range of pump intensities to demonstrate precise control of the cis-state formation yields. The population transfer is resolved crystallographically using quantitative analysis of the occupancy, showing excellent agreement with optical flash-photolysis measurements. The issue of X-ray induced heating, which leads to partial thermal recovery, is also addressed. A temperature increase of 82-112 K is extracted for the fixed-target sample delivery used, which should be accounted for in all future TR-SSX experiments. Finally, the effect of pH on the protonation step is investigated by studying a RSFP mutant with a destabilised ground state conformation. Structural and spectroscopic evidence is presented for a new class of reaction at low pH, involving a cis-neutral/trans-cation isomerisation, in contrasts to the typical cis-anion/trans-neutral reaction at neutral pH. These steady-state observations expand our understanding of acid/base equilibria in the photoreactions of RSFPs.Open Acces