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）：心力衰竭是由于心脏在肌丝水平上的激活或失活而受损。当前的教条表明，CA2+与CTNC结合的心肌收缩，该肌肉在薄丝（TF）中调节了一个“ ON”过程，导致Crossbridge（XB）附着以产生力。心脏放松受到CA2+从CTNC的快速解离触发的TF中的反向“关闭”过程调节。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循环动力学之间的结构动力学之间的关系改变。但是，以前的整体研究未能定义TF过程与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Ca2+诱导的动态相互作用，包括TF蛋白接口处的多种可逆结构变化。这些向后和向后的结构过渡代表了调节XB循环的TF开关过程的谨慎信号传导步骤。根据我们最近的体外动力学研究的发现，我们假设这些向后和向后过渡的微观动力学在构象状态下的微观动力学决定了构象种群之间的平衡关系，并且可能可调节，因此可以提供与CTNC和慢速XB XB Cycligslics Ca2+交流的快速动力学之间的联系。但是，单个步骤的显微镜率常数不能轻松地取决于我们当前的策略，这些策略依赖于集体平均测量值，这些测量掩盖了集合中存在的蛋白质动力学的空间和时间不均匀性。单分子光谱具有独特的优势，即阐明合奏样品固有的这种空间和时间异质性。因此，该项目的总体目的是通过进一步表征TF构象种群之间的平衡关系，探索使用单分子福斯特福斯特福斯特共振能量转移（SMFRET）方法来定义CA2+ - 信号和XB循环之间的动力学联系。重要的是，将获取每个Ca2+诱导的TF结构过渡的显微镜向前或向后转变率常数。将使用SMFRET技术来检验我们的假设：（1）检查CTNC的构象种群在单个重构监管单位的水平上和（2）单分子级别的CTNC构型之间的平衡关系，确定与每个CA2+CAC2+诱导的c逆转录c c c c c c c c c c c c c c c c c c ctni contni contni contni contni contni contni contni contni contni contni ctnc之间的平衡关系。该项目的结果将在解决当前的TF控制XB循环动力学的监管作用问题时至关重要，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从我们的合奏研究和肌肉纤维研究中垂直提高知识。