Structural Kinetics of Thin Filament Regulation at Single Molecule Level

单分子水平细丝调节的结构动力学

• 批准号: 8690957
• 负责人: WEN-JI DONG
• 金额: $ 17.8万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690957
• 关键词: Actins; Address; Binding; Cardiac; Contracts; Depressed mood; Dimensions; Dissociation; Energy Transfer; Equilibrium; Feedback; Fluorescence Anisotropy; Fluorescence Spectroscopy; Head; Heart; Heart failure; Heterogeneity; In Vitro; Individual; Kinetics; Knowledge; Measurement; Microfilaments; Microscopic; Molecular; Molecular Conformation; Muscle Contraction; Muscle Fibers; Muscle relaxation phase; Myocardial; Myocardium; Myosin ATPase; Outcome; Pathology; Play; Population; Preparation; Process; Protein Dynamics; Proteins; Regulation; Relaxation; Research; Role; Sampling; Sarcomeres; Series; Signal Transduction; Skin; Spectrum Analysis; Structural Protein; Techniques; Testing; Thick Filament; Thin Filament; Troponin; Troponin C; Work; base; improved; insight; interest; novel; novel strategies; public health relevance; reconstitution; response; single molecule; success

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.

描述（由申请人提供）：心力衰竭由心肌丝水平的心脏激活或失活受损引起。目前的教条表明，心肌收缩后，Ca 2+结合到cTnC，调节“上”过程中的细丝（TF），导致横桥（XB）连接，以产生力。心脏舒张由TF中的反向“关闭”过程调节，TF中的反向“关闭”过程由Ca 2+与cTnC的快速解离触发。因此，据信这些结构变化的动力学调节XB循环的动力学，使得病理可能由TF的结构动力学和XB循环动力学之间的关系的改变引起。然而，之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TF调节相关的主要特征是TF蛋白之间的Ca 2+诱导的动态相互作用，包括TF蛋白界面处的多个可逆结构变化。这些前向和后向结构转变表示调节XB循环的TF切换过程的离散信令步骤。基于我们最近的体外动力学研究的结果，我们假设，这些正向和反向转变的微观动力学的构象状态决定构象种群之间的平衡关系，是可调的，并因此可能提供的快速动力学之间的联系Ca 2+交换与cTnC和XB循环的缓慢动力学。然而，个别步骤的微观速率常数不能很容易地确定我们目前的策略，依赖于合奏平均测量模糊的空间和时间的不均匀性的蛋白质动力学合奏。单分子光谱学具有独特的优势，解开这种空间和时间的异质性固有的合奏样品。因此，该项目的总体目标是探索使用单分子福斯特共振能量转移（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Ca 2+信号传导和XB循环之间的动力学联系。重要的是，将获得每个Ca 2+诱导的TF结构转变的微观向前或向后转变速率常数。将采用smFRET技术实现两个特定目的以检验我们的假设：（1）在单个重构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，确定与单个重构调控单元内cTnI C结构域的每个Ca 2+诱导可逆结构转变相关的微观速率常数。这个项目的结果将是至关重要的，在解决当前的问题的TF在控制XB循环动力学的调节作用。我们希望从我们提出的单分子研究中获得的信息将有助于垂直推进从我们的合奏研究和肌纤维研究中获得的知识。