Ultrafast time-resolved protein dynamics using X-ray free electron laser crystallography and optical lasers.

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The van Thor group at Imperial College London have previously developed methods based on ultrafast spectroscopy and ultrafast crystallography for temporally resolving the ultrafast dynamics of photoactive-proteins [1, 2]. These methods have allowed dynamic processes occurring in light-sensitive systems to be studied at an unprecedented temporal resolution (between 100fs - 3 ps [3]). The goal of this PhD research is to conduct experiments using these methods to elucidate ultrafast structural behaviour. The initial area of focus will be the development of oriented single protein-crystal measurements. Currently it is possible to perform transient absorption spectroscopy using polarized pump-probe beams [4]. By utilizing the dichroic and non-linear-optical properties of protein crystals, information about the excited state relaxation pathways can be inferred. A novel deployment of this methodology would be to investigate wave-mixing phenomena in protein crystals. The secondary area of focus for this project will involve investigation of the excited state dynamics of a specific family of photoswitching pigments known as phytochrome (Phy). Phytochromes are involved in light detection within plants. They are key in regulating plant's growth and maintaining their development [5]. The photocycle of Phy is known to convert between a dark-adopted Pr state and a photoswitched Pfr state [6] with a 25% yield after excitation to the S1 excited state [7]. This is prior to a Franck Condon (FC) band decay with a lifetime around 150 fs [6, 7]. This project will then aim to perform an ultrafast time-resolved experiment to 'image' the FC motion after excitation. Specifically the technique of time-resolved serial femtosecond crystallography (TR-SFX) will be utilised, probing with an X-ray free electron laser (XFEL) such as the Stanford linac coherent light source (LCLS) or Spring-8 angstrom compact free electron laser (SACLA). Variation in the pump wavelength and timing with respect to the XFEL-probe would allow for different snapshots of the FC motion to be captured, building up a novel animation of the overall relaxation process. To enable such an experiment, firstly, high-resolution (1 Å) crystals will be developed to enable the chosen phytochrome to be resolved at an atomic scale resolution. Characterisation of the photocycle for these crystals would then be performed using flash-photolysis and transient absorption measurements at the Imperial laboratory. Once characterised, improvements in SFX signal-to-noise ratios will be made before carrying out the experiments. [1] DOI: 10.1080/0144235X.2017.1276726[2] DOI: 10.1364/OE.21.008357[3] DOI: 10.1126/science.aad5081[4] DOI: 10.1038/ncomms13977[5] DOI: 10.1073/pnas.1403096111[6] DOI: 10.1074/jbc.M114.571661[7] DOI: 10.1073/pnas.0812056106[8] DOI: 10.1002/lpor.200710005