SINGLE-MOLECULE DYNAMICS OF TARGET BINDING BY CALMODULIN

描述：(逐字摘自申请者的摘要)这项工作的总体目标 该项目是检测单个钙调蛋白(CaM)分子的功能。钥匙 关于Cam如何识别和绑定目标的问题需要回答。 CaM是许多生化钙信号通路中的关键环节。灵活的 凸轮的结构和各种各样的潜在目标表明 分子水平上存在构象分布。单分子 因此，为了研究构象，人们提出了实验 CaM和CaM-靶复合体的涨落和非均质性。鉴于 传统方法测量总体平均、单分子测量 探测结构和靶标激活的个体差异。初步 描述了发现了绑定的分布的调查 质膜钙调素结合区的构象 CA-ATPase(PMCA)。有人认为，这种分布与 酶活性水平的分布，提供了一种机制 酶调节的微调。这项提议旨在检验这一假设。 该提议还试图了解靶标的分子机制。 通过检测单个绑定事件进行识别和绑定。是这样的 探测目标需要测量。拟议的研究还将 测量PMCA中CaM结合和酶激活之间的偶联， 允许对这一机制进行详细调查。迈出的重要一步 该项目将开发在观察过程中固定蛋白质的方法 实现单分子结合和激活的荧光探针。 因为钙调蛋白调节众多的生物过程，所以这项工作是 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。一种理解 条件下钙调蛋白与靶蛋白相互作用的研究 涉及细胞信号或氧化应激将是设计中的关键 缓解涉及这些过程的疾病的治疗方法。实验性的 方法将结合最近开发的单分子技术 荧光共振能量转移和定点定向光谱学 突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.