Ultrafast Dynamics at Protein Interfaces

蛋白质是大而复杂的分子，在生命系统的几乎所有过程中起着关键作用。它们令人难以置信的多样化作用包括通过结合病毒和细菌来保护细胞，读取DNA以构建新分子，携带信息以协调细胞功能，构建细胞结构并最终将分子储存和传递到需要它们的位置。所有这些过程的共同点是蛋白质需要与其环境相互作用，必须在其界面上相互作用。因此，毫不夸张地说，了解蛋白质界面对了解蛋白质功能至关重要。这项研究方案的主要目的是发展这样一种认识。表征蛋白质界面的一个问题是，它们在分子水平上极不均匀，包括带电、中性、氢键和疏水残基，所有这些残基与（通常）水环境的相互作用都不同。表征这种环境需要分子比例的探针，因此可用的工具非常有限。第二个问题是，界面是一个非常动态的环境，所以分子尺度探针确实需要时间分辨结构变化的能力，这些变化可能发生在从纳秒到秒的巨大时间尺度范围内。唯一符合要求的工具是荧光分子探针，因为荧光是一种对环境非常敏感的功能，并且具有允许亚纳秒观察的自然时间尺度。一个潜在的缺点是，荧光分子探针的加入可能足以扰乱人们试图研究的局部结构。我们的解决方案是使用唯一的强荧光氨基酸，色氨酸，作为荧光探针。荧光数据的测量和分析所需的方法在我们的实验室已经发展得很好。通过研究超过100飞秒（十亿分之一秒）分辨率的时间依赖性荧光，我们可以提取关于荧光分子位置的结构和动力学的非常详细的信息。为了将这些方法扩展到色氨酸荧光的研究，我们将使我们的光谱仪适应紫外激发和检测的需要。然后，我们将设计一个肽样品的层次结构，从几个残基的特定序列到单个α螺旋，直到具有已知的二级和三级结构的完整蛋白质。通过这种方式，我们将能够控制单个色氨酸的环境。因此，我们将探索色氨酸位点的动力学，作为其局部结构、极性、电荷和溶剂可及性的函数。最后，我们将通过加入已知与蛋白质界面相互作用的分子来修饰介质，并研究它们对动力学的影响。通过这样的研究，我们将建立一个蛋白质-水界面动力学的全面图景。当然，这种实验必须辅以理论分析。我们的结果将为蛋白质界面的计算机模拟提供严格的测试和刺激。通过这种方式，我们将对这个至关重要的环境有一个完整的了解。

Proteins are large and complex molecules that play a key role in almost all processes in living systems. Their incredibly diverse roles include protecting the cell by binding viruses and bacteria, reading DNA to build new molecules, carrying the messages to coordinate cell functions, building the structures of the cell and finally storing and delivering molecules to the sites where they are needed. What all these processes have in common is the need for the protein to interact with its environment, necessarily at its interface. Thus it is no exaggeration to say that understanding the protein interface is essential to understanding protein function. This research programme is primarily aimed at developing such an understanding.One problem in characterising protein interfaces is that they are extremely inhomogeneous at the molecular level, comprising charged, neutral, H-bonding and hydrophobic residues, all of which interact differently with the (usually) aqueous environment. To characterise such an environment requires a probe of molecular proportions, so the available tools are very limited. A second problem is that the interface is a very dynamic environment, so the molecular scale probe really requires the ability to time resolve structure changes which may occur on a huge range of timescales from nanoseconds to seconds. The only tools which fit the bill are fluorescent molecular probes, since fluorescence is a very sensitive function of the environment and has a natural timescale that permits subnanosecond observations. One potential disadvantage is that the addition of a fluorescent molecular probe can be sufficient to perturb the local structure one is trying to study. Our solution to this is to use the only strongly fluorescent amino acid, tryptophan, as the fluorescence probe. The methods required for the measurement and analysis of fluorescence data are already well developed in our laboratory. By studying the time dependent fluorescence with better than 100 femtosecond (one hundred million billionths of a second) resolution we can extract very detailed information about the structure and dynamics of the site of the fluorescent molecule. To extend these methods to the study of tryptophan fluorescence we will adapt our spectrometer for UV excitation and detection required. We will then design a hierarchy of peptide samples ranging from specific sequences of a few residues through individual alpha helices up to complete proteins with known secondary and tertiary structure. In this way we will be able to control the environment of the single tryptophan. Thus we will probe dynamics at the tryptophan site as a function of the local structure, its polarity, its charge and its solvent accessibility. Finally we will modify the medium by incorporating molecules which are known to interact with the protein interface, and investigate their effect on the dynamics. By such studies we will build up a comprehensive picture of dynamics at the protein - aqueous interface. Of course such experiments must be complements by theoretical analysis. Our results will provide both a severe test of and a stimulus too computer simulations of the protein interface. In this way we will develop a complete picture of this vital environment.