Pulsed laser light source for time-resolved fluorescence spectroscopy

膜蛋白是生物体中许多过程的关键组分。因此，彻底了解这些大分子的功能对于解释复杂系统（如我们的中枢神经系统）至关重要。近年来，X射线晶体学、EPR和荧光光谱学帮助解决了许多结构-功能关系，从而更好地理解与其功能相关的蛋白质构象变化。然而，人们对蛋白质在原子尺度上的实际动力学行为知之甚少。蛋白质的运动是以何种方式发生的，以及哪些物理定律可以用来描述它们，这仍然是未知的。使用荧光或电生理方法从系综测量获得的蛋白质动力学描述了蛋白质的初始和活化状态之间的分布，但没有提供关于运动本身的信息。出现的一个问题是运动发生的速度有多快，因为这将提供有关过渡过程的能量景观的信息。例如，在电压门控离子通道中，门控电流-由门控电荷位移引起-在毫秒范围内测量。然而，从噪声分析，已经建立了5微秒的过渡时间的上限。蛋白质折叠的速度甚至更快。本研究的目的是使用荧光和磷光寿命测量，以确定上限和下限的过渡速度，并调查其敏感性的组成周围的蛋白质。这些知识将使我们能够根据蛋白质的晶体结构更准确地预测蛋白质的功能，并帮助我们更直接地操纵它们的功能，以设计具有特定特征的结构。

Membrane proteins are key components for many processes in the organism. Therefore, a thorough understanding of the functioning of these macromolecules is of utmost importance for the explanation of complex systems such as our central nervous system. In recent years, x-ray crystallography, EPR- and fluorescence spectroscopy have helped to solve many structure-function relationships so that the conformational changes of the proteins related to their function are better understood. Nevertheless, little is known about the actual dynamic behavior of the proteins on an atomic scale. It remains unknown in which manner the movements of the proteins occur and which physical laws can be used to describe them. Protein dynamics obtained from ensemble measurements using fluorescence or electrophysiological methods describe the distribution between the initial and the activated state of the protein, but provide no information about the movement itself. One question that arises is how fast the movements occur since this would offer information about the energy landscape of the transition process. For instance in voltage-gated ion channels, gating currents - caused by gating charge displacements - are measured in the millisecond range. Nevertheless from noise analysis, an upper limit of 5 microseconds for the transition time has been established. Protein folding is known to occur even faster. The aim of this study is to use fluorescence and phosphorescence lifetime measurements to determine upper and lower limits for the transition speed and investigate their sensitivity to the composition of the surrounding protein. This knowledge will enable us to predict with higher certainty the function of proteins based on their crystal structure and will help us to manipulate their function more directed in order to engineer constructs with defined characteristics.