Pulsed laser light source for time-resolved fluorescence spectroscopy

用于时间分辨荧光光谱的脉冲激光光源

• 批准号: 330906-2006
• 负责人: Blunck, Rikard
• 金额: $ 7.18万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2006
• 资助国家: 加拿大
• 起止时间: 2006-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=325981
• 关键词: Pulsed; laser; light; source; time

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.

膜蛋白是生物体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我们的中枢神经系统等复杂系统是至关重要的。近年来，X射线结晶学、电子顺磁共振和荧光光谱学已经帮助解决了许多结构-功能关系，以便更好地了解与其功能相关的蛋白质的构象变化。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以什么方式发生的，以及哪些物理定律可以用来描述它们。通过使用荧光或电生理方法的集合测量获得的蛋白质动力学描述了蛋白质初始状态和激活状态之间的分布，但没有提供关于运动本身的信息。出现的一个问题是，移动发生的速度有多快，因为这将提供有关过渡进程的能源格局的信息。例如，在电压门控离子通道中，由门控电荷位移引起的门控电流在毫秒范围内测量。然而，根据噪声分析，已确定过渡时间的上限为5微秒。众所周知，蛋白质折叠发生得更快。这项研究的目的是使用荧光和磷光寿命测量来确定转换速度的上限和下限，并调查它们对周围蛋白质组成的敏感性。这一知识将使我们能够基于蛋白质的晶体结构更确定地预测蛋白质的功能，并将帮助我们更直接地操纵蛋白质的功能，以便设计出具有明确特征的结构。