FLUORESCENCE STUDY OF TRP IN CHEY AFTER T-JUMP

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们建议用色氨酸荧光和T-JUMP技术研究Chey与Chey结合结构域膜相对应的16个残基的复合体。当Chey未被磷酸化时，肽残基与Chey之间的亲和力较弱，而在磷酸化状态下，其亲和力约为Chey的25倍。发现单一色氨酸的内源性色氨酸荧光在多肽结合时被猝灭，这一信号损失可用于在动力学实验中监测结合。利用以前的核磁共振，所提出的机制可能涉及主要的非活性构象和能够结合多肽的少数活性构象之间的异构化。在这个观点中，磷酸化的作用是改变活性形式和非活性形式之间的平衡。我们建议使用激光T跳跃技术来监测静态和磷酸化状态下的结合动力学。我们还建议研究活性构象和非活性构象之间平衡发生变化的突变体集合。

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.