Ultrafast chemical biology in the gas phase

自从人类整个基因序列被阐明以来，科学家们就被蛋白质结构的丰富信息淹没了。然而，尽管蛋白质结构的可用性是有价值的，但它可能不提供关于特定蛋白质的生物功能的任何信息。在光诱导生物分子过程中，生色团的蛋白质环境对决定生色团的反应途径和产物分布起着至关重要的作用。在基于溶液的反应中，生色团分子在溶剂中自由移动，而在基于蛋白质的反应中，蛋白质环境对生色团中组成原子的运动提供了静态和动态的约束。为了详细了解蛋白质环境在光诱导生物过程中的作用，有必要研究受控环境中发色团的动力学，即气相中的发色团。实验挑战之一是在其受控的蛋白质环境中产生稳定的发色团来源。电喷雾电离(ESI)已经成为一种非常强大的软电离方法，它能够将存在于溶液中的任何分子在其自然环境或控制良好的人工环境中带入气相，包括大至几百千道尔顿的蛋白质。这种动力学发生在核重排的时间尺度上，即飞秒时间尺度(1飞秒=十亿分之一秒)。因此，飞秒激光光源是实时观察这些生物过程动力学的理想工具。但任何时间分辨的飞秒实验的价值都取决于探测方案，而挑战是找到一种全局的探测方法。时间分辨光电子成像光谱学是最近出现的一种非常有效的技术，用于绘制气相中的超快动力学过程。初始的泵浦激光脉冲激发发色团中的电子，延迟的探测激光脉冲电离分子。光电子的动能及其角分布提供了有关电离时生色团的几何形状及其电子波函数的信息。电离是一种全球现象，因此使用这种方法，原则上对可以调查的系统类型没有限制。我们建议设计、建造和优化一种独特的仪器，包括电喷雾或纳米喷射源、飞行时间质谱仪和光电子成像设备，用于研究数百kDa的真实生物系统的超快动力学。为了测试该仪器，我们将研究模型生物系统细菌视紫红质的动力学--该分子负责视觉过程中的光检测。尽管这项科学本身将是有趣的，因为关于光化学途径仍然存在一些争议，但该项目最成功的结果将是证明这一新仪器作为研究基本生物过程的通用工具的潜力。从长远来看，我们希望探索使用这种方法来研究瞬时蛋白质-蛋白质相互作用、蛋白质-配体相互作用和基本蛋白质折叠机制的可能性。然后，我们将处于有利地位，可以探索突变蛋白质环境或塑造飞秒光脉冲的可能性，从而使我们能够识别特定生物系统特有的动态行为模式。

Since the elucidation of the entire human genetic sequence, scientists have been inundated with a wealth of information on protein structure. However, although the availability of a protein structure is valuable, it may not provide any information on the biological function of a specific protein. In photoinduced biomolecular processes, the protein environment of the chromophore plays an essential role in determining the reaction pathway and product distribution of the chromphore. Whilst in solution based reactions the chromophore molecules move freely around within the solvent, in protein based reactions the protein environment provides both a static and dynamical constraint on the motions of the constituent atoms within the chromophore. In order to understand the role of the protein environment on photoinduced biological processes in detail it is necessary to investigate the dynamics of the chromophore in a controlled environment, i.e. in the gas-phase. One of the experimental challenges is generating a stable source of the chromophore in its controlled protein environment. Electrospray ionisation (ESI) has emerged as a very powerful soft ionisation method that has the capability of taking any molecule that exists in solution into the gas phase in its native environment or a well-controlled artificial environment, including proteins as large a several hundred kilodaltons. The dynamics take place on the timescale of nuclear rearrangement, i.e. the femtosecond timescale (1 femtosecond = 1 millionth of a billionth of a second). Therefore, femtosecond laser sources are ideal tools for observing the dynamics of these biological processes in real time. But the value of any time-resolved femtosecond experiment depends on the probe scheme and the challenge is to find a global detection method. Time-resolved photoelectron imaging spectroscopy has recently emerged as an extremely powerful technique for mapping out ultrafast dynamical processes in the gas phase. An initial pump laser pulse excites an electron in the chromophore and a delayed probe laser pulse ionises the molecule. The kinetic energies of the photoelectrons and their angular distributions provide information about the geometry of the chromophore and its electronic wave function at the time of ionisation. Ionisation is a global phenomenon so with this approach there is, in principle, no limitation to the type of system that that can be investigated. We propose to design, construct and optimise a unique instrument comprising an electrospray or nanospray source, a time-of-flight mass spectrometer and photoelectron imaging apparatus for investigating the ultrafast dynamics of real biological systems of several hundreds of kDa. To test the instrument we will investigate the dynamics of the model biological system Bacteriorhodopsin - the molecule responsible for light detection in the process of vision. Although the science will be interesting in its own right as there is still some controversy over the photochemical pathway, the most successful outcome of this project will be to demonstrate the potential of this new instrument as a generic tool for studying fundamental biological processes. In the longer term we would hope to explore the possibilities of using this type of approach to study transient protein-protein interactions, protein-ligand interactions and fundamental protein folding mechanisms. We would then be in a strong position to explore the possibility of mutating the protein environment or shaping the femtosecond light pulses to enable us to identify patterns of dynamic behaviour unique to specific biological systems.