Nanomechanics of the extracellular matrix

描述(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，它提供了机械支架，指导器官和组织的发育并保持其完整性。细胞外基质中的蛋白质受到各种机械力的作用，每个分子可以达到数百个pN。然而，我们对受力下的蛋白质动力学的了解仍然有限，因为蛋白质的纳米力学不能用溶液生物化学来研究。在纳米尺度上的单分子力谱研究表明，在拉伸力的响应下，蛋白质经历了提供弹性反应的去折叠/重折叠循环，同时揭示了涉及机械信号转导的隐蔽结合部位。力还会产生其他后果，比如改变化学反应的速度。虽然这些现象的存在现在已经被很好地记录下来，但对蛋白质在力的作用下的动态的分子水平的理解仍然是缺乏的。在过去这笔赠款的资助期内，我们开发了力钳光谱学技术。这项技术直接测量蛋白质中机械反应的力依赖性。机械反应的力依赖性可以与过渡态结构直接相关，过渡态结构决定了反应的速度。机械过渡状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的方法来测量几个重要的力学反应的力依赖性，例如折叠、展开和小亲核分子对二硫键的还原。这些实验将研究与细胞外基质的弹性和信号传递有关的关键蛋白质的机械展开过渡状态：纤维连接蛋白和Talin，以及模型蛋白质，如泛素和I27免疫球蛋白。一旦拉力减小，这些蛋白质就会迅速折叠，关闭它们的机械力化学信号。然而，人们对蛋白质如何在压力下折叠知之甚少。在这里，我们将使用各种力钳制方案来识别和表征这些蛋白质在其各自的机械折叠轨迹中所经历的各个阶段。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：在力的作用下二硫键的减少。我们将使用我们灵敏的力钳分析来研究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。拟议中的实验将揭示支配暴露在机械力下的蛋白质动力学的结构的分子细节，这对于理解蛋白质弹性和细胞外基质中的机械力化学信号至关重要。 与公共健康相关：健康和患病组织的弹性由蛋白质对机械应力的反应方式决定。因此，了解蛋白质的纳米力学性质具有重要的医学意义。力钳光谱学现在提供了涉及蛋白质机械响应的关键结构的详细视图。这些研究提供了对蛋白质在所有生物体中弹性组织组装中所扮演的角色的基本了解。

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.