Nanomechanics of the extracellular matrix

描述（由申请人提供）：细胞外基质是由蛋白质和多糖组成的复杂网络，提供机械支架，指导发育并保持器官和组织的完整性。细胞外基质的蛋白质暴露在广泛的机械力下，可以达到每个分子数百pN。然而，我们对力作用下蛋白质动力学的理解仍然有限，因为蛋白质的纳米力学不能用溶液生物化学来研究。纳米尺度的单分子力光谱研究表明，在拉伸力的作用下，蛋白质经历展开/再折叠循环，提供弹性响应，同时揭示了参与机械信号转导的隐结合位点。力也有其他后果，比如改变化学反应的速率。虽然这些现象的存在现在已经被很好地记录下来，但对蛋白质在力作用下的动力学的分子水平的理解仍然缺乏。在过去的资助期间，我们开发了力钳光谱技术。这项技术直接测量了蛋白质中机械反应的力依赖性。机械反应的力依赖性与过渡态结构直接相关，过渡态结构决定了反应的速率。机械过渡态决定了蛋白质在受到拉伸力时的行为。在这里，我们建议使用这些新方法来测量几个重要的机械反应的力依赖性，如折叠、展开和小亲核试剂对二硫键的还原。这些实验将检查参与细胞外基质弹性和信号传导的关键蛋白质的机械展开过渡状态：纤维连接蛋白，talin和模型蛋白，如泛素和I27免疫球蛋白蛋白。当拉力降低时，这些蛋白质迅速折叠，关闭它们的机械化学信号。然而，人们对蛋白质在外力作用下如何折叠知之甚少。在这里，我们将使用各种力夹协议来识别和表征这些蛋白质在其单独的机械折叠轨迹中所访问的各个阶段。我们还将研究控制细胞外基质蛋白质弹性和折叠的另一个关键过程；二硫键在作用力下还原。我们将使用我们的灵敏力夹法来检查施加在蛋白质上的机械拉伸力如何改变二硫键还原的化学机制。提出的实验将揭示在机械力作用下支配蛋白质动力学的结构的分子细节，这对于理解细胞外基质中的蛋白质弹性和机械化学信号至关重要。

DESCRIPTION (provided by applicant): The extracellular matrix is a complex web of proteins and polysaccharides that provides the mechanical scaffold that guides the development and preserves the integrity of organs and tissues. The proteins of the extracellular matrix are exposed to a broad range of mechanical forces that can reach into the hundreds of pN per molecule. However, our understanding of protein dynamics under force remains limited because the nanomechanics of proteins cannot be studied using solution biochemistry. Single molecule force-spectroscopy studies, at the nanometer scale, have demonstrated that in response to a stretching force, proteins undergo unfolding/refolding cycles providing for an elastic response while uncovering cryptic binding sites involved in mechanical signal transduction. Force also has other consequences such as altering the rate of chemical reactions. While the existence of these phenomena is now well documented, a molecular level understanding of the dynamics of proteins under force is still lacking. During the past funding period of this grant we developed the force-clamp spectroscopy technique. This technique directly measures the force-dependency of mechanical reactions in proteins. The force-dependency of a mechanical reaction can be directly related to the transition state structure, which determines the rate of the reaction. The mechanical transition state determines how a protein will behave when exposed to a stretching force. Here we propose to use these novel approaches to measure the force dependency of several important mechanical reactions such as folding, unfolding and the reduction of disulfide bonds by small nucleophiles. These experiments will examine the mechanical unfolding transition states of crucial proteins involved in the elasticity and signaling of the extracellular matrix: fibronectin, and talin and model proteins such as ubiquitin and the I27 immunoglobulin protein. Upon a reduction in the pulling force, these proteins rapidly fold, turning off their mechano-chemical signals. However, very little is known of how proteins fold under force. Here we will use a variety of force-clamp protocols to identify and characterize the individual stages visited by these proteins during their individual mechanical folding trajectories. We will also study another critical process governing the elasticity and folding of proteins of the extracellular matrix; disulfide bond reduction under force. We will use our sensitive force-clamp assay to examine how a mechanical stretching force applied to a protein alters the chemical mechanisms of disulfide bond reduction. The proposed experiments will uncover the molecular details of the structures that dominate the dynamics of proteins exposed to mechanical forces, crucial to understanding protein elasticity and mechano-chemical signaling in the extracellular matrix. PUBLIC HEALTH RELEVANCE: The elasticity of healthy and diseased tissues is determined by how proteins respond to mechanical stress. It is then of great medical importance to understand the nanomechanical properties of proteins. Force-clamp spectroscopy now provides a detailed view of the key structures involved in the mechanical responses of proteins. These studies provide a fundamental understanding of the roles played by proteins in the assembly of elastic tissues in all organisms.