Single molecule studies of titin elasticity

描述(由申请人提供)：这项建议的长期目标是了解肌动蛋白弹性及其调节的分子机制。肌动蛋白是一种巨大的模块化蛋白质，通过363个外显子的选择性剪接，编码各种各样的异构体，决定了人体内数百种肌肉类型的弹性。心脏肌动蛋白的主要异构体是一种3-丙二醛蛋白，其对机械负荷的动态反应由弹性I带区域的两大类模块决定：单独折叠的免疫球蛋白样区(Ig)以及弹性体区PEVK和N2B。我们开创了多蛋白工程和单分子AFM研究这些模块的机械性能的先河，成功地生成了Titin弹性的粗粒度视图。然而，在我们早期的实验中，单个蛋白质分子的力和长度同时变化，阻碍了对Titin弹性的完整定量描述的发展。为了解决这一缺点，我们开发了一种新的力钳光谱技术，它可以监测在恒定力下持有的蛋白质的长度动态。与彻底改变离子通道研究的电压钳技术类似，力钳技术现在可以更深入、更详细地研究TiTiN中的力相关反应。例如，这些新技术现在允许我们研究机械力对硫醇/二硫键交换的影响，这是调节Titin和许多其他蛋白质中二硫键的常见化学反应。我们将使用这些方法来研究硫氧还蛋白酶的力依赖活性，硫氧还蛋白是一种通过二硫键还原而调节细胞氧化还原状态的普遍存在的酶。这些研究不仅将揭示调节Titin弹性的新机制，还将有助于开发一种新的结构生物学技术，该技术可以探测催化过程中的酶动力学，分辨率达到亚埃，这是目前任何其他方法都无法实现的。力钳技术还允许详细检查推动延伸蛋白质崩溃的分子作用力，延伸蛋白质是肌动蛋白弹性的最关键成分，其来源仍未解决。我们将使用力钳方案结合溶剂替代来研究Titin的PEVK、N2B和未折叠的Ig结构域的反弹力。这些实验将检验这一假设，即未折叠的Titin模块的弹性主要由延伸的多肽的疏水塌陷决定，挑战了当前的Titin延伸性范式。最后，通过力钳光谱的使用，我们将重新定义机械稳定性的概念，以定量比较TiIn Ig结构域，首先检测TiIn N末端结构域Z1和Z2的力学性质。然后，我们将检验Z1Z2-端粒素复合体在反平行Titin分子之间形成机械稳定锚的假设，并进一步探索该复合体中突变与人类疾病有关的机械作用。这项拟议的研究将研究Titin弹性的基本分子机制，并开发一种新形式的蛋白质光谱来跟踪Angstrom尺度下的蛋白质动力学。与公共健康相关的肌肉弹性是由巨大的蛋白质Titin设定的。因此，了解这种蛋白质的机械性质具有重要的医学意义。研究蛋白质力学需要在纳米尺度上使用一套新的工具来研究单个Titin蛋白质如何对机械力做出反应。这些研究将允许开发正常和疾病状态下组织(如心肌)弹性的机械模型。

DESCRIPTION (provided by applicant): The long term aim of this proposal is to understand the molecular mechanisms underlying titin elasticity and its regulation. Titin is a giant modular protein that through alternative splicing of 363 exons, codes for a wide variety of isoforms that determine the elasticity of the hundreds of muscle types found in the human body. The predominant isoform of cardiac titin is a 3 MDa protein whose dynamic response to mechanical load is determined via two broad classes of modules in the elastic I band region: individually folded immunoglobulin like (Ig) domains and the elastomeric regions PEVK and N2B. We have pioneered polyprotein engineering and single molecule AFM studies of the mechanical properties of these modules, successfully generating a coarse grained view of the elasticity of titin. However, in our earlier experiments the force and length of single protein molecules varied simultaneously, preventing the development of a complete quantitative description of titin elasticity. To address this shortcoming we have developed the new force-clamp spectroscopy technique that monitors the length dynamics of a protein held under a constant force. Similar to the voltage-clamp techniques that revolutionized the study of ion channels, force-clamp now permits a deeper and more detailed study of force-dependent reactions in titin. For example, these new techniques now permit us to study the effect of mechanical forces on thiol/disulfide exchange, the common chemical reaction that regulates disulfide bonding in titin and in many other proteins. We will use these methods to study the force-dependent activity of the enzyme thioredoxin, a ubiquitous regulator of cellular redox states via disulfide bond reduction. These studies will not only uncover novel mechanisms of regulating titin elasticity but will also help develop a new structural biological technique that can probe enzyme dynamics during catalysis, with sub-Angstrom resolution, currently not possible with any other method. Force-clamp techniques also permit a detailed examination of the molecular forces driving the collapse of an extended protein, the most crucial component of titin elasticity whose origin remains unresolved. We will use force-clamp protocols combined with solvent substitution to study the recoil forces of the PEVK, N2B and of the unfolded Ig domains of titin. These experiments will test the hypothesis that the elasticity of unfolded titin modules is determined mainly by hydrophobic collapse of the extended polypeptides, challenging current paradigms of titin extensibility. Finally, through the use of force-clamp spectroscopy we will redefine the concept of mechanical stability to quantitatively compare titin Ig domains, first examining the mechanical properties of the titin N-terminus end domains Z1 and Z2. We will then test the hypothesis that the Z1Z2-telethonin complex forms a mechanically stable anchor between antiparallel titin molecules, and further probe the mechanical role of mutations in this complex linked to human disease. The proposed studies will examine the fundamental molecular mechanisms underlying titin elasticity, as well as develop a new form of protein spectroscopy to track protein dynamics in the Angstrom scale. PUBLIC HEALTH RELEVANCE Muscle elasticity is set by the giant protein titin. It is then of great medical importance to understand the mechanical properties of this protein. Studying protein mechanics has necessitated a novel set of tools at the nanoscale to study how single titin protein respond to a mechanical force. These studies will permit the development of mechanistic models of the elasticity of tissues such as heart muscle, in normal and diseased states.