Single molecule studies of titin elasticity

描述（由申请人提供）：巨蛋白titin负责心肌弹性，在人体血液循环中起关键作用。心脏舒张时左心室的充血，以及随后通过主动脉将血液排出的能力，都取决于titin的弹性。心肌的弹性在体内受氧化还原信号的调控，并受基因突变的修饰。事实上，titin基因的基因突变被发现是扩张型心肌病（一种最常见的心脏病）的主要原因。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个巨大的35000个氨基酸长的titin蛋白的弹性，这仍然是一个艰巨的挑战。Titin在机械负荷下在体内工作，这使得用经典的蛋白质生物化学技术研究它变得困难，极大地限制了我们对这种蛋白质功能的理解。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通过氧化还原信号调节titin弹性的分子基础。我们将检验一氧化氮信号通过修改弹性i带的埋藏半胱氨酸残基来增加titin弹性的假设，这些残基在titin被机械拉伸时暴露出来。我们还将研究氧化应激触发的假设

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.