Single Molecule Studies of Titin Elasticity

描述(申请人提供)：我们使用单分子技术来研究巨型肌肉蛋白titin的机械设计。Titin的长度是肌肉肌节的一半，可以超过一微米长。肌动蛋白与肌节I带重叠的区域决定了肌肉的弹性。I带titin具有独特的模块化设计，由免疫球蛋白(Ig)类结构域的串联重复组成，中间有一个富含P、E、V和K残基的区域，对于心肌来说，另一个区域由名为N2B的独特序列组成。我们的长期目标是了解正常和疾病状态下肌动蛋白弹性的分子机制。 对Titin组成表达区域的几个模块的详细单分子研究揭示了一种复杂的依赖于结构的机械设计。然而，Titin的弹性是通过大量令人震惊的外显子对其I带的选择性剪接进行精细调节的，其中106个外显子编码Ig模块，114个外显子编码PEVK序列。目前还不知道肌动蛋白选择性剪接区编码的机械基序，也不知道它们是如何控制肌动蛋白弹性的。此外，大量的Titin Ig结构域被证明具有形成机械稳定的二硫键的潜力。此外，Titin分子被证明是相互作用的，这表明每个弹性细丝可能由几个Titin分子组成，这些分子可能形成弹性四元结构。因此，Titin的弹性可能受到几种新机制的精细调节。 我们结合单分子AFM和蛋白质工程技术来研究Titin机制的各个组成部分。我们将检查不同Ig模块的机械特性和Titin选择性剪接区的PEVK序列。我们将研究二硫键的形成对Ig结构域的力学性质的影响。我们将研究其他蛋白质折叠的机械稳定性，例如肌肉蛋白质utroin的螺旋拓扑结构和高度保守的蛋白质泛素的α-β拓扑结构。我们将研究它们机械设计的起源，并将其与Titin免疫球蛋白模块进行比较。通过使用蛋白质工程和诱变，我们将研究Pro(以及E、V和K残基)在控制PEVK区域弹性方面所起的作用。利用不同的寡聚结构域，我们将组装基于Titin的多蛋白束，以研究超分子排列对Titin力学的影响。我们希望，除了揭示调节Titin弹性的新机制外，我们的研究还将有助于进一步发展单分子力谱研究的新领域。

DESCRIPTION (provided by applicant): We use single molecule techniques to study the mechanical design of the giant muscle protein titin. Titin spans half the length of a muscle sarcomere and can be over a micrometer long. The region of titin that overlaps with the sarcomeric I band determines muscle elasticity. I band titin has a characteristic modular design composed of tandem repeats of immunoglobulin (Ig) type domains, interrupted by a region rich in P, E, V and K residues and, in the case of cardiac muscle, another region made of a unique sequence named N2B. Our long-term aim is to understand the molecular mechanisms underlying titin elasticity in normal and diseased states. Detailed single molecule studies of a few modules of the constitutively expressed regions of titin have revealed a complex structure-dependent mechanical design. However, titin elasticity is finely regulated through alternative splicing of its I band by a stunning number of exons, 106 exons coding for Ig modules and 114 exons coding for PEVK sequences. Nothing is known of the mechanical motifs encoded by the alternatively spliced regions of titin or how they govern titin elasticity. Also, a large number of titin Ig domains have been shown to have a potential for the formation of mechanically stabilizing disulfide bridges. Moreover, titin molecules have been shown to interact suggesting that each elastic filament may be composed of several titin molecules, which may form elastic quaternary structures. Hence, titin elasticity might be finely modulated by several novel mechanisms. We combine single molecule AFM and protein engineering techniques to study the individual building blocks of titin mechanics. We will examine the mechanical features of the different Ig modules and the PEVK sequences in the alternatively spliced regions of titin. We will examine the role of disulfide bridge formation on the mechanical properties of Ig domains. We will examine the mechanical stability of other protein folds such as the helical coiled-coil topology of the muscle protein utrophin and the alpha-beta topology of the highly conserved protein ubiquitin. We will study the origin of their mechanical design and compare it to that of the titin immunoglobulin modules. Through the use of protein engineering and mutagenesis, we will examine the role played by prolines (as well as E, V and K residues) in controlling the elasticity of the PEVK regions. Using a variety of oligomerization domains we will assemble bundles of titin based polyproteins to examine the effect of supra-molecular arrangements on titin mechanics. We expect that in addition to uncovering new mechanisms of regulating titin elasticity, our studies will contribute to further develop the new field of research on single molecule force spectroscopy.