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

伦敦帝国理工学院的货车托尔小组先前已经开发了基于超快光谱学和超快晶体学的方法，用于在时间上分辨光敏蛋白质的超快动力学[1，2]。这些方法允许以前所未有的时间分辨率（100 fs- 3 ps [3]）研究光敏系统中发生的动态过程。这项博士研究的目标是使用这些方法进行实验，以阐明超快结构行为。最初的重点领域将是定向单蛋白质晶体测量的发展。目前，可以使用偏振泵浦-探测光束进行瞬态吸收光谱[4]。通过利用蛋白质晶体的二向色性和非线性光学性质，可以推断有关激发态弛豫途径的信息。这种方法的一个新的部署将是研究蛋白质晶体中的波混合现象。该项目的第二个重点领域将涉及一个特定的光开关色素家族（光敏色素）的激发态动力学的研究。光敏色素参与植物体内的光检测。它们是调节植物生长和维持其发育的关键[5]。已知Phy的光循环在暗采用Pr状态和光开关Pfr状态之间转换[6]，在激发到S1激发态后产率为25%[7]。这是在Franck Condon（FC）带衰减之前，寿命约为150 fs [6，7]。然后，该项目将旨在执行超快时间分辨实验，以“成像”激励后的FC运动。具体而言，将利用时间分辨连续飞秒晶体学（TR-SFX）技术，用X射线自由电子激光器（XFEL）进行探测，如斯坦福大学直线加速器相干光源（LCLS）或Spring-8埃紧凑型自由电子激光器（SACLA）。相对于XFEL探针的泵浦波长和定时的变化将允许捕获FC运动的不同快照，从而建立整个弛豫过程的新颖动画。为了实现这样的实验，首先，将开发高分辨率（1 μ m）晶体，以使所选择的光敏色素能够以原子级分辨率分辨。这些晶体的光循环特性将在帝国实验室使用闪光光解和瞬态吸收测量进行。一旦特征化，在进行实验之前，将对SFX信噪比进行改进。[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

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