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

使用无 X 射线电子激光晶体学和光学激光器的超快时间分辨蛋白质动力学。

• 批准号: 1961372
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1961372
• 关键词: Ultrafast; time; resolved; protein; dynamics

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

伦敦帝国理工学院的范索尔小组此前开发了基于超快光谱和超快结晶学的方法，用于在时间上解析光活性蛋白质的超快动力学[1，2]。这些方法使人们能够以前所未有的时间分辨率(100fs-3ps[3])研究发生在光敏系统中的动态过程。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晶蛋白质测量的发展。目前，使用偏振泵浦探测光束进行瞬时吸收光谱是可能的[4]。利用蛋白质晶体的二色性和非线性光学性质，可以推断出有关激发态弛豫路径的信息。这一方法的一个新部署将是研究蛋白质晶体中的波混合现象。这个项目的第二个重点领域将包括研究一种特定的光开关颜料家族的激发态动力学，即光敏色素(Phy)。光敏色素参与植物体内的光探测。它们是调节植物生长和维持其发育的关键。已知Phy的光周期在暗吸收的Pr态和光开关的PfR态之间转换[6]，在激发到S1激发态之后有25%的产率[7]。这是在弗兰克康登(FC)带衰变之前的，它的寿命约为150飞秒[6，7]。然后，这个项目的目标是进行超快的时间分辨实验，以便在激励后对FC运动进行‘成像’。具体地说，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(XFEL)探测，例如斯坦福直线加速器相干光源(LCLS)或Spring-8 Angstrom紧凑型自由电子激光器(SACLA)。泵浦波长和定时相对于XFEL探测器的变化将允许捕获FC运动的不同快照，构建整体弛豫过程的新颖动画。为了能够进行这样的实验，首先，将开发高分辨率(1？)晶体，以使所选的光敏色素能够在原子尺度上被分辨。然后将在帝国实验室使用闪光光解和瞬时吸收测量来表征这些晶体的光循环。一旦确定了特征，将在进行实验之前改进SFX信噪比。[1]DOI：10.1080/0144235X.2017.1276726[2]DOI：10.1364/OE.21.008357[3]DOI：10.1126/cience.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