FLUORESCENCE STUDY OF TRP IN CHEY AFTER T-JUMP

T-JUMP 后 Chey 中 TRP 的荧光研究

• 批准号: 7373146
• 负责人: Frederick W. Dahlquist
• 金额: $ 0.27万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373146
• 关键词: FLUORESCENCE; STUDY; TRP; CHEY; AFTER

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to study the complex of CheY with a 16 residue peptide corresponding to the CheY binding domain FliM using tryptophan fluorescence and T-jump technology. The affinity between the peptide residue and CheY is weak when CheY is unphosphorylated and about 25 times stronger in the phosphorylated state. The intrinsic tryptophan fluorescence of the single Trp was found to be quenched on peptide binding, a signal loss that could be exploited to monitor binding in a kinetic experiment. Using NMR previously, a suggested mechanism may involve an isomerization between a predominant inactive conformation and a minority active conformation that is able to bind the peptide. In this view, the role of the phosphorylation is to shift the equilibrium between the active and inactive forms. We propose to monitor the binding kinetics using the laser T-jump technique in both resting and phosphorylated states. We are also proposing to investigate a collection of mutants with altered equilibria between the active and inactive conformation.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。我们建议使用色氨酸荧光和 T 跳跃技术研究 CheY 与对应于 CheY 结合域 FliM 的 16 个残基肽的复合物。当 CheY 未磷酸化时，肽残基与 CheY 之间的亲和力较弱，而在磷酸化状态下则强约 25 倍。发现单个色氨酸的内在色氨酸荧光在肽结合时被猝灭，这种信号损失可用于监测动力学实验中的结合。之前使用 NMR，建议的机制可能涉及主要的非活性构象和能够结合肽的少数活性构象之间的异构化。按照这种观点，磷酸化的作用是改变活性和非活性形式之间的平衡。我们建议使用激光 T 跳跃技术在静息状态和磷酸化状态下监测结合动力学。我们还建议研究一组活性和非活性构象之间平衡发生改变的突变体。