Nanomechanics of the extracellular matrix

描述（由申请人提供）：细胞外基质是蛋白质和多糖的复杂网络，其提供引导器官和组织发育并保持其完整性的机械支架。细胞外基质的蛋白质暴露于大范围的机械力，其可以达到每分子数百pN。然而，我们对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使用溶液生物化学进行研究。单分子力谱研究，在纳米尺度上，已经证明，在响应于拉伸力，蛋白质经历展开/重折叠循环提供弹性响应，同时发现神秘的结合位点参与机械信号转导。力还有其他的作用，比如改变化学反应的速率。虽然这些现象的存在现在是有据可查的，分子水平的理解下的蛋白质动力学的力量仍然缺乏。 在过去的资助期间，我们开发了力钳光谱技术。该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的方法来测量力的依赖性的几个重要的机械反应，如折叠，展开和减少二硫键的小亲核试剂。这些实验将检查参与细胞外基质的弹性和信号传导的关键蛋白质的机械展开过渡状态：纤连蛋白和talin以及模型蛋白质如泛素和I27免疫球蛋白。当拉力减小时，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，我们将使用各种力钳协议，以确定和表征这些蛋白质在其各自的机械折叠轨迹访问的各个阶段。我们还将研究另一个决定细胞外基质蛋白质的弹性和折叠的关键过程;二硫键在外力下的还原。我们将使用我们灵敏的力钳分析来研究施加到蛋白质上的机械拉伸力如何改变二硫键还原的化学机制。拟议的实验将揭示结构的分子细节，这些结构主导着暴露于机械力的蛋白质的动力学，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。 公共卫生相关性：健康和患病组织的弹性取决于蛋白质对机械应力的反应。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一个详细的视图的关键结构参与的机械反应的蛋白质。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起作用的基本理解。

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.