Pulsed laser light source for time-resolved fluorescence spectroscopy

膜蛋白是生物体中许多过程的关键成分。因此，彻底了解这些大分子的功能对于解释像我们的中枢神经系统这样的复杂系统至关重要。近年来，x射线晶体学、EPR-和荧光光谱已经帮助解决了许多结构-功能关系，从而更好地了解蛋白质与其功能相关的构象变化。然而，人们对蛋白质在原子尺度上的实际动态行为知之甚少。目前尚不清楚蛋白质的运动是如何发生的，也不知道可以用哪些物理定律来描述它们。利用荧光或电生理方法从集合测量中获得的蛋白质动力学描述了蛋白质的初始状态和激活状态之间的分布，但没有提供有关运动本身的信息。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源格局的信息。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，从噪声分析来看，已经确定了过渡时间的上限为5微秒。蛋白质折叠的速度甚至更快。本研究的目的是利用荧光和磷光寿命测量来确定过渡速度的上限和下限，并研究它们对周围蛋白质组成的敏感性。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，并将帮助我们更有针对性地操纵它们的功能，以便设计具有明确特征的结构。

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.