Time-resolved protein dynamics using x-ray crystallography and optical spectroscopy
File(s)
Author(s)
Baxter, James Michael
Type
Thesis
Abstract
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.
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.
Version
Open Access
Date Issued
2021-03
Date Awarded
2021-08
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
van Thor, Jasper
Sponsor
Engineering and Physical Sciences Research Council
Grant Number
1961372
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)