International Collaboration in Chemistry on Control of Excited State Proton Transfer in GFP

2008年诺贝尔奖表彰了绿色荧光蛋白（GFP）和相关荧光蛋白作为监测细胞和组织中蛋白质定位和运输工具的革命性生物应用。然而，来自水母Aequorea维多利亚的原型GFP在吸收光子后表现出高效（>80%）质子转移方面也是独特的。质子转移有助于大多数生物反应，但通常发生在难以直接观察的时间尺度上。因此，用飞秒光脉冲触发质子转移的能力为理解蛋白质如何控制这一无处不在的过程提供了无与伦比的机会。我们提出的调查建议是假设驱动的，旨在了解超快激发态质子转移（ESPT）反应的动力学和控制。特别是，我们希望确定低频促进振动，形成质子转移的理论治疗的一个基本要素。我们提出了初步测量的低频振动相干发生在ESPT反应的持续时间内。控制ESPT在GFP是目前热烈讨论，我们建议帮助解决悬而未决的问题，如荧光和熵反应障碍，发色团冷却的作用，低频模式识别和分配，以及异质性和蛋白质发色团结构的相互转换。超快动力学，振动相干光谱（Raman），瞬态可见-可见和可见-红外吸收光谱，飞秒受激拉曼光谱（FSRS），和相干控制光谱将用于解剖微观细节。将特别强调温度依赖性研究。进一步发展包括蛋白质环境在内的高水平计算化学计算将有助于解释光谱结果。反过来，实验结果将为计算方法提供必要的校准。

The 2008 Nobel Prize recognized the revolutionary biological applications of the green fluorescent protein (GFP) and related fluorescent proteins as tools for monitoring protein localization and trafficking in cells and tissues. However, the prototype GFP, from the jellyfish Aequorea victoria, is also unique in exhibiting highly efficient (>80%) proton transfer following absorption of a photon. Proton transfer contributes to most biological reactions, but typically takes place on time scales that are difficult to observe directly. The ability to trigger proton transfer with a femtosecond light pulse thus yields unequalled opportunities for understanding how proteins control this ubiquitous process. Our proposed investigations proposal is hypothesis-driven and aims to understand the dynamics and control of the ultrafast excited state proton transfer (ESPT) reaction. In particular, we hope to identify low frequency promoting vibrations that form an essential element of theoretical treatments of proton transfer. We present preliminary measurements of low frequency vibrational coherences that occur within the duration of the ESPT reaction. Control of ESPT in GFP is currently fervidly discussed and we propose to help resolve outstanding issues such as the enthalpic and entropic reaction barriers, the role of chromophore cooling, low frequency mode identification and assignment, and heterogeneity and interconversions of the protein-chromophore structure. Ultrafast kinetics, vibrational coherence spectroscopy (VCS), transient visible-visible and visible-infrared absorption spectroscopy, femtosecond stimulated Raman spectroscopy (FSRS), and coherent control spectroscopy will be used to dissect the microscopic details. Particular emphasis will be placed on temperature dependent studies. Further development of high level computational chemistry calculations that include the protein environment will aid in interpretation of the spectroscopic results. In turn, the experimental results will provide an essential calibration for the computational methods.