PEPTIDE THIOAMIDES AS FLUORESCENCE QUENCHING PROBES TO MONITOR PROTEIN DYNAMICS

肽硫代酰胺作为荧光淬灭探针来监测蛋白质动态

• 批准号: 8362581
• 负责人: Ernest James Petersson
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362581
• 关键词: Acetamides; Complex; DNA Sequence Rearrangement; Fluorescein; Fluorescence; Fluorescence Spectroscopy; Funding; Grant; Image; Laboratories; Lasers; Measures; Modification; Monitor; Motion; National Center for Research Resources; Nature; Optics; Peptides; Physiologic pulse; Principal Investigator; Process; Protein Dynamics; Proteins; Research; Research Infrastructure; Resolution; Resources; Source; Thioacetamide; Thioamides; Time; United States National Institutes of Health; Vertebral column; X-Ray Crystallography; chromophore; cost; flexibility; fluorophore; oxoamide; protein structure; research study

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 当今生物化学家面临的巨大挑战之一是了解折叠和 蛋白质的结构动力学。很明显，通过 X 射线晶体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功能。荧光光谱允许人们在纳秒时间尺度上观察蛋白质运动，但相对较大的荧光团尺寸妨碍了以高分辨率分配这些运动。如果光学探针可以做得足够小，它们就可以提供真正观察和剖析蛋白质运动所需的时间和结构分辨率。为此，我们正在开发硫代酰胺，即肽主链的单原子修饰，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。我们希望使用 RLBL 脉冲二极管激光器装置在 408 nm 处激发吖啶酮和荧光素，并在分子间和分子内猝灭实验中测量草酰胺和硫代酰胺存在下的荧光寿命，其中两个发色团通过刚性 (Pro ）或柔性（Gly/Ser）肽接头。这些实验应该使我们能够确认硫代酰胺猝灭的动态性质。