Photoelectron spectroscopy in a liquid microjet: unravelling the excited state dynamics of photoactive proteins

自然界中高效的光诱导过程的广泛使用激发了人们在功能性合成系统中开发类似过程的努力。例如，荧光蛋白使生物成像发生了革命性变化。然而，我们对发色团周围的蛋白质在光活性蛋白质中的关键作用的理解仍然远未完成。本研究的目的是通过系统地研究一系列发色团在真空、溶液和蛋白质中的电子结构和激发态动力学，从分子水平上了解光活性蛋白质发色团与其环境之间的相互作用。光电子能谱是测量分子中电子结合能的一种特别有价值的工具，飞秒时间分辨光电子能谱已经成为探测光激发后分子中能量流动的一种非常强大的技术。在飞秒时间分辨光电子能谱中，飞秒激光脉冲(泵浦)激发分子，延迟一段时间后，第二个飞秒激光脉冲(探头)电离分子。由此产生的光电子的动能和角分布提供了有关分子在电离时的电子和振动状态的信息。在不同的泵浦-探测延迟下记录一系列光电子光谱，使我们能够记录分子中电子和振动能量流动的分子“电影”。时间分辨光电子能谱在研究气相和固体中的电子结构和动力学方面已被证明是非常成功的，但水溶液则是一个更大的挑战。液体微喷技术的最新技术进步使溶液中的时间分辨光电子能谱成为一种真实而令人兴奋的可能性。我们将利用这些发展创造一种光电子能谱装置，能够研究发色团在溶液和蛋白质环境中的飞秒动力学。在生物系统中，光激发后的分子动力学由生色团的分子和电子结构以及它与环境的相互作用所控制。例如，GFP发色团的环境决定了它的光学性质：发色团在其桶状蛋白质中是强荧光的，而当蛋白质变性时荧光消失，但复性后又恢复；分离的发色团在水溶液中是非荧光的，在气相中也是非荧光的，但分离分子在气相中的吸收光谱与蛋白质中的吸收光谱非常相似。为了揭示环境在确定荧光蛋白质的光学性质中的重要作用，我们将研究发色团的电子和结构性质的系统性变化以及蛋白质的突变如何影响光激发后的结合能和电子弛豫。高水平的电子结构和动力学计算将有助于解释实验结果。将使用有机化学和分子生物学方法来创建一系列发色团和蛋白质，用于系统地评估电子、结构和环境变化的影响。已经组建的多学科小组非常适合解决这一将在许多科学领域产生影响的重要问题。

The extensive use of efficient light-induced processes in nature is inspiring efforts to exploit similar processes in functional synthetic systems. For example, fluorescent proteins have revolutionalised biological imaging. However, our understanding of the crucial role of the protein surrounding the chromophore in photoactive proteins is still far from complete. The aim of this research is to gain a molecular level understanding of the interactions between photoactive protein chromophores and their environment by a systematic investigation of the electronic structure and excited state dynamics of a series of chromophores in vacuo, in solution and in protein. Photoelectron spectroscopy is a particularly valuable tool for measuring the binding energies of electrons in molecules and femtosecond time-resolved photoelectron spectroscopy has emerged as a very powerful technique for probing the flow of energy in a molecule following photoexcitation. In femtosecond time-resolved photoelectron spectroscopy, a femtosecond laser pulse (pump) excites a molecule and after some delay a second femtosecond laser pulse (probe) ionises the molecule. The kinetic energy and angular distribution of the resulting photoelectrons provides information about the electronic and vibrational states of the molecule at the time of ionisation. Recording a series of photoelectron spectra at different pump-probe delays allows us to record a molecular "movie" of the flow of electronic and vibrational energy in the molecule. Time-resolved photoelectron spectroscopy has proved remarkably successful for investigating electronic structure and dynamics in the gas phase and in solids but aqueous solutions have presented more of a challenge. Recent technical advances in liquid microjet technology have enabled time-resolved photoelectron spectroscopy in solutions to become a real and exciting possibility. We will exploit these developments to create a photoelectron spectroscopy apparatus that is capable of investigating the femtosecond dynamics of chromophores in solution and in their protein environments. In a biological system, the molecular dynamics after photoexcitation are controlled by the molecular and electronic structure of the chromophore and by its interaction with the environment. For example, the environment of the GFP chromophore defines its optical properties: the chromophore is strongly fluorescent inside its barrel-shaped protein, while the fuorescence is lost when the protein is denatured but it returns upon renaturation; the isolated chromophore is non-fluorescent in aqueous solution and it is also non-fluorescent in the gas phase, yet the absorption spectrum of the isolated molecule in the gas phase is remarkably similar to that in the protein. In order to unravel the important role of the environment in defining the optical properties of fluorescent proteins, we will investigate how systematic changes to the electronic and structural properties of the chromophore and mutations to the protein influence binding energies and electronic relaxation following photoexcitation. High-level electronic structure and dynamics calculations will assist the interpretation of the experimental results. Organic chemistry and molecular biology methods will be employed to create the series of chromophores and proteins for systematic evaluation of the influence of electronic, structural, and environmental changes. The multidisciplinary team that has been assembled is ideally suited to tackle this important problem which will have an impact in many areas of science.