PEPTIDE THIOAMIDES AS FLUORESCENCE QUENCHING PROBES TO MONITOR PROTEIN DYNAMICS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 当今生物化学家面临的最大挑战之一是理解折叠和 蛋白质的结构动力学。很明显，通过X射线结晶学获得的蛋白质结构的静态图像通常不足以进行机械表征。许多蛋白质需要复杂的结构重排才能发挥作用。荧光光谱学使人们能够在ns时间尺度上观察蛋白质的运动，但相对较大的荧光团大小排除了在高分辨率下指定这些运动的可能性。如果光学探测器足够小，它们可以提供必要的时间和结构分辨率来真正观察和剖析蛋白质运动。为此，我们正在开发硫代酰胺，即单原子修饰的多肽骨架，作为极小的荧光猝灭探针。我们用乙酰胺或硫代乙酰胺对几种荧光团进行了Stern-Volmer猝灭实验。在分子间和分子内猝灭实验中，我们希望使用RLBL脉冲二极管激光装置在408 nm激发吖啶酮和荧光素，并测量在草酰胺和硫代酰胺存在下的荧光寿命，在分子间和分子内猝灭实验中，这两个发色团被刚性(Pro)或柔性(Gly/Ser)肽连接体分开。这些实验应该能让我们确认硫代酰胺猝灭的动力学性质。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. One of the great challenges facing biochemists today is to understand the folding and structural dynamics of proteins. It is clear that the static images of protein structure obtained by X-ray crystallography are often insufficient for mechanistic characterization. Many proteins require complex structural rearrangements for function. Fluorescence spectroscopy allows one to observe protein motions on the ns timescale, but the relatively large size of fluorophores precludes assigning these motions at high resolution. If optical probes could be made sufficiently small, they could provide the time and structural resolution necessary to truly observe and dissect protein motions. To this end, we are developing thioamides, single atom modifications of the peptide backbone, as extremely small fluorescence quenching probes. We have carried out Stern-Volmer quenching experiments of several fluorophores with acetamide or thioacetamide. We would like to use the RLBL pulsed diode laser setup to excite acridone and fluorescein at 408 nm and measure fluorescent lifetimes in the presence of oxoamides and thioamides, both in intermolecular and intramolecular quenching experiments in which the two chromophores are separated by either rigid (Pro) or flexible (Gly/Ser) peptide linkers. These experiments should allow us to confirm the dynamic nature of thioamide quenching.