Spectroscopy and Instrumentation Core

仪器光谱核心（SIC）D2的目标有两个方面：首先，在D2.2中详细说明 计划的发展方向，是改进现有的工具，开发新的工具，或 本发明提供了旨在改善信噪比、特异性和时间分辨率的方法和程序， 光谱技术，并将其与功能技术，如电生理学相结合。的 第二个目标（详见MPSDC的D2.4组件）是为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 目前安装的技术。 将在两个主要发展领域开展工作。电子顺磁共振（EPR） 荧光提供了关于膜蛋白结构变化动力学的补充信息。 时间分辨EPR技术，如快速冷冻淬火（EPR）将是一个优先事项， 开发基于微流体的微流控装置。同样，我们计划利用大范围的 时间尺度探测荧光。我们将通过优化探针和完善检测来实现这一目标 技术（包括系综和单分子），使膜中的动态过程的跟踪 proteins.虽然单荧光团的有限光子通量和光稳定性通常限制了单分子 成像技术到ms制度，我们将通过开发 分子内稳定的有机荧光团。 这些总目标将通过一系列具体项目来实现： 目的1：进一步发展和完善微流控快速冷冻淬灭EPR系统， 通过快速混合反应物产生的冷冻样品的测量，并使样品易于获得 对财团成员。这项技术将与双电子-电子 共振（DEER）和电子核双共振（ENDOR）实验。 目的2：进一步发展和增强单分子荧光技术： a）测试、扩大和提供高性能有机荧光团。测试那些开发的 在核心D1中加入了非天然氨基酸技术 B）建立一个将磁镊与单分子荧光相结合的装置。这种技术将 用于对膜蛋白施加力，以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）开发多色单分子FRET设置，其将允许检测同步或相关的荧光信号。 在多个域之间的运动。 d）开发具有增强的时间分辨率的测量单分子荧光的装置，以快速分辨 构象变化 目的3：进一步发展和加强整体荧光技术： a）改进远程环境试验装置，并使联合体成员能够在下列地点测量距离： 功能性膜蛋白 B）开发一种装置，以测量微秒时间尺度中的纳秒荧光团寿命， 电生理学 c）用新设计的光电探测器-电压转换器改进荧光探测系统 并开发一个新的更强大的采集系统，将用于所有上述设置。

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.