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 s 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.
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