Single molecule studies of titin elasticity

描述(申请人提供)：巨大的蛋白质titin负责心脏肌肉的弹性，在人类的血液循环中发挥关键作用。人心脏舒张期左心室的血液充盈，以及随后通过主动脉排出血液的能力，取决于肌动蛋白的弹性。在体内，心肌的弹性由氧化还原信号调节，并受基因突变的影响。事实上，Titin基因的基因突变被发现是扩张型心肌病的主要原因，扩张型心肌病是最常见的心脏病之一。我们还远未了解氧化还原信号或突变如何改变人类心脏中这种由35,000个氨基酸组成的巨型Titin蛋白的弹性，这仍然是一个巨大的挑战。Titin在体内是在机械负荷下工作的，这使得用经典的蛋白质生物化学技术来研究它变得困难，极大地限制了我们对这种蛋白质如何发挥作用的了解。在这里，我们的目标是使用最先进的单分子力谱技术来确定氧化还原信号调节Titin弹性的分子基础。我们将检验这一假说，即一氧化氮信号通过改变当Titin被机械拉伸时暴露的弹性I带的埋藏的半胱氨酸残基来增加Titin的弹性。我们还将检验氧化应激触发 相反，通过形成埋藏的二硫键使肌动蛋白变硬，限制了蛋白质的延展性。我们将利用蛋白质组学技术来鉴定从人类心脏组织中提纯的天然肌动蛋白分子中针对b氧化还原修饰的残留物。因此，我们的建议旨在确定参与体内肌动蛋白弹性生理调节的关键残基，以及它们的作用方式。我们将应用我们的发现来开发第一个完整的人体心脏肌动蛋白弹性计算模型。这个模型将基于我们目前对Titin的物理和化学知识。我们的目标是找出控制Titin力学性质的所有关键残基，然后基于蒙特卡罗和布朗动力学建立基因-表型数学模型，预测蛋白质对不同类型的生理或病理刺激的弹性反应，例如模拟运动或脑梗塞的刺激。我们将用氧化还原信号的已知效应，以及与心肌疾病相关的肌动蛋白突变的不断增长的基因组数据库来验证这一模型。这种将肌动蛋白弹性的物理学与其通过细胞信号进行化学调节的综合以前从未尝试过，并将提供第一个定量工具来揭示许多肌肉组织疾病的病因学。这个模型的一个高度发达的版本可以用来评估被发现有titin基因突变的人类心肌表型的严重程度。

DESCRIPTION (provided by applicant): The giant protein titin is responsible for the elasticity of heart muscle, playing a key role in the circulation of blood in humans. The filling with blood o the left ventricle of the human heart during diastole, and its subsequent ability to eject it throuh the aorta, is dependent on the elasticity of titin. The elasticity of heart muscle is regulated in ivo by redox signaling and modified by genetic mutations. Indeed, genetic mutations of the titin gene were found to be a primary cause of dilated cardiomyopathies, one of the most common forms of heart disease. We are far from understanding how redox signaling or mutations can alter the elasticity of this giant 35,000-amino-acid long titin protein in the human heart, which remains a formidable challenge. Titin operates in vivo under a mechanical load, making it difficult to study it with classical protein biochemistry techniques, greatly limiting our understanding of how this protein functions. Here we aim to use state-of-the-art single molecule force spectroscopy techniques to identify the molecular basis of regulation of titin elasticity by redox signaling. We will examine the hypothesis that nitric oxide signaling increases titin elasticity by modifying buried cysteine residues of the elastic I-band that become exposed when titin is mechanically stretched. We will also examine the hypothesis that oxidative stress triggers the opposite effect, by stiffening titin through the formation of buried disulfide bonds that limitthe extensibility of the protein. We will utilize proteomics techniques to identify residues targeted b redox modifications in native titin molecules purified from human cardiac tissue. Thus, our proposal aims to identify the key residues involved in the physiological regulation of titin elasticity in vivo, and their mode of action. We will apply our findings to develop the first full computational model of human cardiac titin elasticity. This model will be based on our current knowledge of the physics and chemistry of titin. Our aim is to identify all the key residues controlling the mechanical properties of titin, and then implement a gene-to-phenotype mathematical model based on Monte Carlo and Brownian Dynamics, to predict the elastic response of the protein to different types of physiological or pathological stimuli, such those mimicking exercise or infarction. We will validate this model with the known effects of redox signaling, and with a growing genomic database of titin mutations associated with heart muscle disease. Such a synthesis of the physics of titin elasticity with its chemical regulation by cellulr signaling has never been attempted before, and will provide the first quantitative tool to uncover the etiology of numerous muscle tissue diseases. A highly developed version of this model may be used to evaluate the severity of heart muscle phenotypes in humans that are discovered to have mutations in the titin gene.