Molecular Dissection of Calmodulin Domain Functions

描述（由申请人提供）：为了了解调节蛋白复合物的功能重要状态，必须直接测量驱动其组装和配体诱导构象反应的热力学和动力学力。钙调蛋白（Calmodulin， CAM）是一种重要的真核钙传感器，是一种变构单体，通过与靶蛋白的钙依赖性关联，控制神经传递、肌肉收缩、生育和代谢等元素。历史上，它被认为有两种功能状态：“开”（饱和4个钙离子）或“关”（载脂蛋白，无钙）。一些靶蛋白被认为可以逆转这种逻辑，并使用载脂蛋白形式的CaM作为激活剂。它的两个同源结构域（N和C）被认为是与靶蛋白相关的等效伙伴。然而，CaM的两个EF-hand结构域现在被认为在一些靶标的激活中具有可分离的作用。本提案的主要目的是阐明CaM中控制其生理作用的域特异性转变的分子机制。本实验室对2类调节离子通道缺陷的草履虫CaM突变体的研究表明：(a)螺旋B和C的突变降低了热稳定性，使钙结合更有利；(B)螺旋a和D之间的相互作用是离子结合和柔韧性物种差异的关键决定因素；(C) N和C结构域的共价偶联加剧了它们的差异。所研究的所有CaM突变体都能够在载脂蛋白和钙饱和条件下结合靶肽，这表明调节失败是通过中间状态的改变途径发生的（即有缺陷的构象反应或降低的结合常数）。该研究计划将(a)确定结构域间连接子对N-和c -结构域特性的作用，以及(b)定量评估突变PCaM与选定靶蛋白之间的相互作用。离子和目标结合的构象转换和能量学将使用异核磁共振、荧光、CD、质谱和流体动力学方法（超离心、色谱）来确定。这种对CaM的分析将有助于理解结构域相互作用的途径以及这些高度同源的结构域在靶活化中所起的生理不同作用。这将有助于更好地理解钙水平的同步变化如何调节真核生物的各种生理过程。

DESCRIPTION (provided by applicant): To understand the functionally significant states of a regulatory protein complex, one must directly measure the thermodynamic and kinetic forces that drive its assembly and ligand-induced conformational responses. Calmodulin (CAM), an essential eukaryotic calcium sensor, is an allosteric monomer that controls elements of neurotransmission, muscle contraction, fertility and metabolism through its calcium-dependent association with target proteins. Historically, it was viewed as having two functional states: "on" (saturated with 4 calcium ions) or "off" (apo, calcium-free). A few target proteins were recognized to reverse this logic and use the apo form of CaM as an activator. Its two homologous domains (N & C) were believed to be equivalent partners in association with target proteins. However, the two EF-hand domains of CaM are now recognized to have separable roles in activation of some targets. The major goal of this proposal is to elucidate molecular mechanisms of domain-specific transitions in CaM that govern its physiological roles. Studies by this laboratory of 2 classes of Paramecium CaM mutants, defective in regulating ion channels, demonstrated that (a) mutations in helices B & C that lower thermostability make calcium binding more favorable, (b) interactions between helices A & D are key determinants of species differences in ion binding and flexibility and (c) covalent coupling of the N- and C-domains exacerbates their differences. All of the CaM mutants studied were able to bind target peptides under both apo and calcium-saturating conditions, demonstrating that regulatory failure is occurring via altered pathways through the intermediate states (i.e., defective conformational responses or reduced binding constants). This research program will (a) determine the roles of the interdomain linker on properties of the N- and C-domain and (b) quantitatively evaluate the interactions between mutant PCaM's and selected target proteins. Conformational switching and energetics of ion and target binding will be determined using heteronuclear NMR, fluorescence, CD, mass spectroscopy, and hydrodynamic methods (ultracentrifugation, chromatography). This analysis of CaM will contribute to understanding pathways of domain interactions and the physiologically distinct roles these highly homologous domains play in target activation. This will lead to a better understanding of how synchronized changes in calcium levels modulate diverse physiological processes in eukaryotes.