Structural Kinetics of Thin Filament Regulation at Single Molecule Level

描述(由申请人提供)：心力衰竭是由于心脏在肌丝水平上的激活或失活受损造成的。目前的学说认为，心肌在钙离子与cTNC结合后收缩，这调节了导致交叉桥(XB)附着的细丝(Tf)中的“开”过程以产生力。心肌松弛是通过快速从cTNC中解离钙离子而触发的Tf中一个反向的“关闭”过程来调节的。因此，人们认为，这些结构变化的动力学调节了XB循环的动力学，因此，病理可能是由于Tf和XB循环动力学的结构动力学之间的关系发生了变化。然而，以前的系综研究未能定义Tf过程和XB循环之间的动力学联系。与转铁蛋白调节相关的一个主要特征是钙离子诱导转铁蛋白之间的动态相互作用，包括转铁蛋白界面的多个可逆结构变化。这些向前和向后的结构转变代表了调节XB循环的TF交换过程中谨慎的信号步骤。根据我们最近的体外动力学研究结果，我们假设构象状态下这些向前和向后转变的微观动力学决定了构象群体之间的平衡关系，并且是可调节的，因此可能提供了钙与cTNC交换的快速动力学和XB循环的缓慢动力学之间的联系。然而，单个步骤的微观速率常数不容易通过我们目前的策略确定，这些策略依赖于集合平均测量，这掩盖了集合中存在的蛋白质动力学的空间和时间不均匀。单分子光谱学具有独特的优势，可以解开整体样品固有的空间和时间异质性。因此，该项目的总体目标是探索使用单分子Forster共振能量转移(SmFRET)方法，通过进一步表征控制Tf构象群体之间转变的平衡关系，来定义钙信号和XB循环之间的动力学联系。重要的是，将获得每个钙诱导的Tf结构转变的微观前向或向后转变速率常数。使用smFRET技术来验证我们的假设有两个特定的目标：(1)在单个重组调控单元水平上检查cTNC构象群体之间的平衡关系；(2)在单分子水平上，确定与每个钙诱导的单个重组调控单元内cTnI C-结构域的可逆结构转变相关的微观速率常数。这个项目的结果对于解决目前Tf在控制XB循环动力学中的调节作用这一问题将是至关重要的。我们期望从我们建议的单分子研究中获得的信息将有助于垂直推进从我们的整体研究和肌肉纤维研究中获得的知识。

DESCRIPTION (provided by applicant): Heart failure results from impaired activation or deactivation of the heart at the level of the myofilament. Current dogma suggests that cardiac muscle contracts upon Ca2+ binding to cTnC, which regulates an "on" process in the thin filament (TF) leading to crossbridge (XB) attachment to generate force. Cardiac relaxation is regulated by a reverse "off" process in the TF triggered by rapid dissociation of Ca2+ from cTnC. It is thus believed that the kinetics of these structural changes modulate the kinetics of the XB cycle, such that pathology may arise from alterations in the relationship between the structural kinetics of the TF and XB cycling kinetics. However, previous ensemble studies failed to define the kinetic linkage between the TF processes and XB cycling. A main feature associated with TF regulation is Ca2+-induced dynamic interactions among the TF proteins, including multiple reversible structural changes at the TF protein interfaces. These forward and backward structural transitions represent the discreet signaling steps of the TF switching process that regulates XB cycling. Based on the findings from our recent in vitro dynamics study, we hypothesize that the microscopic kinetics of these forward and backward transitions in conformational state dictate equilibrium relationships between conformational populations and are tunable, and may thus provide the linkage between the rapid kinetics of Ca2+ exchange with cTnC and slow kinetics of XB cycling. However, the microscopic rate constants of individual steps cannot be easily determined by our current strategies that rely on ensemble-averaged measurements which obscure the spatial and temporal inhomogeneity of the protein dynamics present in the ensemble. Single-molecule spectroscopy has the unique advantage of unraveling this spatial and temporal heterogeneity inherent in ensemble samples. Accordingly, the overall objective of this project is to explore the use of single-molecule Forster Resonance Energy Transfer (smFRET) approaches to define the kinetic linkage between Ca2+-signaling and XB cycling by further characterizing the equilibrium relationships governing transitions between TF conformational populations. Importantly, microscopic forward or backward transition rate constants for each Ca2+-induced TF structural transition will be acquired. Two Specific Aims will be pursued using smFRET techniques to test our hypothesis: (1) examine the equilibrium relationships between conformational populations of cTnC at the level of single reconstituted regulatory units and (2) at the single-molecule level, determine microscopic rate constants associated with each Ca2+-induced reversible structural transitions of the C-domain of cTnI within single reconstituted regulatory units. Outcomes of this project will be of critical importane in addressing the current issue of the regulatory role of the TF in controlling XB cycling kinetics We expect that the information obtained from our proposed single-molecule studies will help to vertically advance the knowledge gained from our ensemble studies and muscle fiber research.