SINGLE-MOLECULE DYNAMICS OF TARGET BINDING BY CALMODULIN

描述：（逐字来自申请人的摘要） 项目是检测单个钙调素（CaM）分子的功能。关键 关于CaM如何识别和结合到靶点的问题需要得到回答。 CaM是许多生化钙信号通路中的关键环节。柔性 CaM的结构和各种各样的潜在靶点表明， 构象的分布存在于分子水平。单分子 因此，提出了实验，以研究构象 CaM和CaM-靶复合物的波动和异质性。而 常规方法测量系综平均，单分子测量 探测结构和目标激活的个体差异。初步 描述了已经揭示了结合分布的调查， 与质膜的CaM结合结构域结合的CaM构象 Ca-ATPase（PMCA）。有人建议，这种分布与一个 酶的活性水平的分布，提供了一种机制， 酶调节的微调。本提案旨在检验这一假设。 该提案还试图了解靶向的分子机制。 通过检测单个结合事件进行识别和结合。等 需要测量来探测目标。拟议的研究还将 测量PMCA中CaM结合和酶活化之间的偶联， 允许对该机制进行详细研究。的重要一步 该项目将开发在观察过程中使蛋白质变性的方法 实现单分子结合和激活的荧光探针。 由于钙调素调节许多生物过程，这项工作是 与广泛的健康问题有关，包括神经传递和 学习、氧化损伤和衰老以及肌肉激活。的理解 钙调素和靶蛋白之间的相互作用， 涉及细胞信号或氧化应激将是至关重要的设计 治疗，以减轻涉及这些过程的疾病。实验 这种方法将联合收割机结合最近开发的单分子 荧光共振能量转移和定点光谱法 诱变作为蛋白质结构和动力学的探针。

DESCRIPTION: (Verbatim from the Applicant's Abstract) The overall goal of this project is to detect single calmodulin(CaM) molecules as they function. Key questions need to be answered about how CaM recognizes and binds to a target. CaM is a key link in many biochemical calcium signaling pathways. The flexible structure of CaM and the wide variety of potential targets suggests that a distribution of conformations exists at the molecular level. Single-molecule experiments are therefore proposed in order to investigate conformational fluctuations and heterogeneity of CaM and CaM-target complexes. Whereas conventional methods measure an ensemble average, single-molecule measurements probe individual variations in structure and target activation. Preliminary investigations are described that have uncovered a distribution of binding conformations for CaM bound to the CaM-binding domain of the plasma membrane Ca-ATPase (PMCA). It is suggested that this distribution correlates with a distribution of activity levels of the enzyme, providing a mechanism for fine-tuning of enzyme regulation. This proposal seeks to test this hypothesis. The proposal also seeks to understand the molecular mechanisms of target recognition and binding by detecting individual binding events. Such measurements are needed to probe targets. The proposed research will also measure the coupling between CaM-binding and enzyme activation in the PMCA, permitting a detailed investigation of this mechanism. An important step in the project will be developing methods to immobilize the protein during observation and implementing fluorescence probes of single-molecule binding and activation. Because calmodulin regulates numerous biological processes, this work is pertinent to a wide range of health concerns, including neurotransmission and learning, oxidative damage and aging, and muscle activation. An understanding of the interaction between calmodulin and target proteins under conditions involving cellular signaling or oxidative stress will be crucial in designing therapies to alleviate disorders involving these processes. The experimental approach will combine recently developed techniques of single-molecule spectroscopy with fluorescence resonance energy transfer and site-directed mutagenesis as a probe of protein structure and dynamics.