REGULATORY MECHANISMS OF CARDIAC MUSCLE

肌肉收缩是由肌动蛋白和肌动蛋白之间的循环相互作用引起的。 肌球蛋白 所产生的力的大小是 肌动蛋白-肌球蛋白接触形成。 脊椎动物中的这种相互作用 横纹肌是由激活剂钙的结合， 肌钙蛋白C（TnC）的NH/2-结构域，这种结合导致一系列 蛋白质结构的变化与薄 这些蛋白质之间相互作用的能量学。 的 由激活剂钙引起的直接分子事件的细节 绑定仍然知之甚少。 建模研究表明， 激活剂钙诱导几个螺旋的实质性重新取向 位于TnC的NH/2-末端结构域，导致“开放” 构象 这种构象反过来又提供了一个特定的位点， TnC与肌钙蛋白I（TnI）的强相互作用。 这种强烈的相互作用 被认为是触发肌动蛋白-肌球蛋白相互作用的原因， 从而激活肌动球蛋白ATP酶和收缩事件。 TnI来自 心肌具有与磷酸化的活性， 蛋白质在NH/2-末端片段，这是没有发现在骨骼 肌钙蛋白I。 虽然磷酸化的位点是已知的， 关于这个片段如何调节细丝中的Ca 2+信号， 调控 本申请解决了钙调节的某些方面， 心肌中的机制。 拟议的工作有四个主要目标。 （一） 第一个是鉴定上的特定残基或螺旋对 由激活剂钙引起的相互相对运动的TnC 约束力 平衡实验将使用荧光 共振能量转移（FRET），以建立这种钙诱导的 构象变化，随后进行瞬态动力学实验，以时间- 解析构象转变。 心肌TnC突变体 心脏-骨骼嵌合体将用于这些实验。 (2)的 钙激活剂与心肌可逆结合动力学机制 TnC和心脏调节系统将使用瞬时 动力学方法 预期这些动力学结果另外 可用于追踪NH/2-结构域中螺旋的运动。 (3)心脏 TnI在NH/2-末端有一个在骨骼肌TnI中不存在的延伸。 我们将研究这个延伸的磷酸化的关系 它的整体构造。 全长重组突变体和 TnI的截短突变体将用于FRET、流体动力学和结合 问题研究 (4)最后一个目标是研究氨基酸之间的相互关系 钙结合环和侧翼螺旋的酸序列 具有结合亲和力和生物活性的TnC的NH/2-结构域。 具有特定氨基酸改变的心肌TnC突变体 序列将被设计并用于这一目标。 拟议的研究 有望推进我们对分子决定因素的认识， 在心肌的细丝调节中很重要。 我们也 预期预期结果可能为理解 在多大程度上钙触发机制发挥作用的异常 心脏功能

Muscle contraction results from cyclic interactions between actin and myosin. The magnitude of developed force is a function of the number actin-myosin contacts that are formed. This interaction in vertebrate striated muscle is regulated by the binding of activator calcium to the NH/2-domain of troponin C (TnC), and this binding results in a series of changes in both the structures of the proteins associated with the thin filament and the energetics of interactions among those proteins. The details of the immediate molecular events resulting from activator calcium binding are still poorly understood. Modeling studies suggest that activator calcium induces substantial reorientations of several helices located in the NH/2-terminal domain of TnC, resulting in an "open" conformation. This conformation in turn provides specific sites for a strong interaction of TnC with troponin I (TnI). This strong interaction is believed to be responsible for triggering the actin-myosin interaction, thus activating actomyosin ATPase and contractile events. TnI from cardiac muscle has an activity which is related to phosphorylation of the protein in the NH/2-terminal segment and which is not found in skeletal muscle TnI. While the sites of phosphorylation are known, little is known about how this segment may modulate Ca2+ signaling in thin-filament regulation. This application addresses certain aspects of the calcium regulatory mechanism in cardiac muscle. The proposed work has four major goals. (1) The first is identification of pairs of specific residues or helices on TnC that move relative to each other resulting from activator calcium binding. Equilibrium experiments will be done using fluorescence resonance energy transfer (FRET) to establish such calcium-induced conformational changes, followed by transient kinetic experiments to time- resolve the conformational transitions. TnC mutants from cardiac muscle and cardiac-skeletal chimeras will be used for these experiments. (2) The kinetic mechanism of reversible binding of activator calcium to cardiac TnC and the cardiac regulatory system will be studied using transient kinetic methods. It is expected that these kinetic results additionally can be used to track movements of helices in the NH/2-domain. (3) Cardiac TnI has an extension at the NH/2-terminus which is absent in skeletal TnI. We will investigate the relationship of phosphorylation of this extension with its overall conformation. Full-length recombinant mutants and truncated mutants of TnI will be used in FRET, hydrodynamic, and binding studies. (4) The last goal will investigate the relationships of amino acid sequences of the calcium-binding loops and flanking helices in the NH/2-domain of TnC with binding affinity and biological activities. Cardiac muscle TnC mutants with specific alterations in amino acid sequences will be designed and used for this goal. The proposed studies are expected to advance our knowledge of the molecular determinants that are important in thin filament regulation of cardiac muscle. We also expect that the anticipated results may provide a basis to understand the extent to which the calcium triggering mechanism plays a role in abnormal cardiac functions.