Spectroscopy and Instrumentation Core

仪器光谱核心(SIC)D2的目标有两个：第一，在D2.2中详细说明 规划的发展方向，是改进现有的仪器，开发新的仪器，或 旨在提高信噪比、特异度和时间分辨率的方法和程序 光谱技术，并将其与电生理学等功能技术相结合。这个 第二个目标，在MPSDC的D2.4部分详细说明，是向MPSD成员提供服务，以及 整个社区，随着新的发展以及光谱和功能 目前已安装的技术。 我们将致力于两个主要领域的发展。电子顺磁共振(EPR)和 荧光为膜蛋白结构变化的动态变化提供了补充信息。 时间分辨EPR技术，如快速冻结淬火(RFQ)将是优先事项，与 基于微流控技术的RFQ装置的研制。同样，我们计划利用大范围的 用荧光探测的时间尺度。我们将通过优化探针和完善检测来做到这一点 能够跟踪膜内动态过程的技术(包括系综和单分子) 蛋白质。而有限的光子通量和单荧光团的光稳定性通常限制了单分子 成像技术发展到ms体制，我们将通过发展 分子内稳定的有机荧光团。 这些总体目标将通过一系列具体项目来实现： 目的1：进一步开发和完善微流控快速冷冻淬火(RFQ)EPR系统，以使 对快速混合反应物产生的冷冻样品的测量，使RFQ可以访问 给财团的成员。这项技术将与双电子-电子结合使用。 共振(Deer)和电子核双共振(Endor)实验。 目标2：进一步发展和加强单分子荧光技术： A)测试、扩大和提供高性能有机荧光团。测试那些已开发的 与非天然氨基酸技术在核心的d1。 B)建立一种将磁性镊子与单分子荧光相结合的装置。这项技术将会 用于对膜蛋白施加力来研究单个分子的构象变化 同时用荧光探针评估它们的功能。 C)开发一种多色单分子FRET装置，使其能够检测同步或相关的 多个域之间的运动。 D)开发一种测量单分子荧光的装置，提高了时间分辨率，以快速分辨 构象变化 目标3：进一步发展和加强集合荧光技术： A)改进LRET设置，并使其可供联盟成员在#年测量距离 功能性膜蛋白。 B)开发一套在微秒时间尺度上测量纳秒荧光团寿命的装置，结合 电生理学。 C)改进荧光检测系统，采用光电探测器-电压转换器的新设计 并开发一种新的、功能更强大的采集系统，用于上述所有设置。

The objective of the Instrumentation Spectroscopy Core (SIC) D2 is two-fold: first, detailed in D2.2 Planned Direction of Development, is to improve presently available instruments, develop new instruments, or methods and procedures aimed at improving on signal to noise ratio, specificity and time resolution of spectroscopic techniques and combining them with functional techniques, such as electrophysiology. The second objective, detailed in D2.4 Component to the MPSDC, is to provide service to the MPSD members, and the community at large, with the new developments as well as with the spectroscopic and functional techniques presently installed. Two major areas of development will be pursued. Electron paramagnetic resonance (EPR) and fluorescence provide complementary information on the dynamics of structural changes in membrane proteins. Time-resolved EPR techniques, such as rapid freeze quench (RFQ) will be a priority, together with the development of a microfluidic-based RFQ apparatus. Likewise, we plan to take advantage of the large range of time scales probed by fluorescence. We will do so by optimizing the probes and perfecting the detection techniques (both ensemble and single-molecule) that enable the tracking of dynamic processes in membrane proteins. While the finite photon flux and photostability of single-fluorophores typically limits single-molecule imaging techniques to the ms regime, we will push this boundary to the µs regime through the development of intramolecularly stabilized organic fluorophores. These general goals will be carried out through a series of specific projects: AIM 1: To further develop and perfect a microfluidic rapid freeze quench (RFQ) EPR system to enable measurements of frozen samples that are generated by rapid mixing of reactants and make RFQ accessible to members of the consortium. This technique will be applied in conjunction with double electron-electron resonance (DEER) and Electron nuclear double resonance (ENDOR) experiments. AIM 2: To further develop and enhance single-molecule fluorescence techniques: a) Test, expand and make available high-performance organic fluorophores. Test those that are developed with unnatural amino acid technologies in core D1. b) Establish a setup that combines magnetic tweezers with single-molecule fluorescence. This technique will be used to apply force to membrane proteins to study conformational changes on individual molecules while assessing their functionalities with fluorescent probes. c) Develop a multi-color single-molecule FRET setup that will allow detection of synchronized or correlated motions among multiple domains. d) Develop a setup to measure single-molecule fluorescence with enhanced time resolution to resolve fast conformational changes AIM 3: To further develop and enhance ensemble fluorescence techniques: a) Improvement of an LRET setup and make it available to members of the consortium to measure distances in functional membrane proteins. b) Develop a setup to measure nanosecond fluorophore lifetimes in the microsecond time scale combined with electrophysiology. c) Improve the fluorescence detection system with a new design of the photodetector-to-voltage transducer and develop a new more powerful acquisition system that will be used for all of the above setups.