Single Molecule Studies of Titin Elasticity

描述（由申请人提供）：我们使用单分子技术研究巨肌蛋白肌联蛋白的机械设计。肌联蛋白跨越肌肉肌节长度的一半，可以超过一微米长。肌联蛋白与肌节I带重叠的区域决定肌肉弹性。I带肌联蛋白具有由免疫球蛋白（IG）型结构域的串联重复序列组成的特征性模块设计，其被富含P、E、V和K残基的区域中断，并且在心肌的情况下，另一个区域由称为N2 B的独特序列组成。我们的长期目标是了解肌联蛋白在正常和疾病状态下弹性的分子机制。 对肌联蛋白组成性表达区域的几个模块的详细单分子研究揭示了复杂的结构依赖性机械设计。然而，肌联蛋白的弹性通过其I带的选择性剪接被数量惊人的外显子精细地调节，106个外显子编码IG模块，114个外显子编码PEVK序列。对于肌联蛋白可变剪接区编码的机械基序或它们如何支配肌联蛋白弹性，我们一无所知。此外，大量肌联蛋白IG结构域已被证明具有形成机械稳定二硫键的潜力。此外，肌联蛋白分子已经显示出相互作用，这表明每个弹性细丝可以由几个肌联蛋白分子组成，其可以形成弹性四级结构。因此，肌联蛋白的弹性可能是精细调制的几个新的机制。 我们结合联合收割机单分子原子力显微镜和蛋白质工程技术来研究肌联蛋白力学的各个组成部分。我们将研究不同的IG模块和在肌联蛋白的可变剪接区的PEVK序列的机械特征。我们将研究二硫键形成对IG结构域的机械性能的作用。我们将研究其他蛋白质折叠的机械稳定性，如肌肉蛋白utrophin的螺旋卷曲螺旋拓扑结构和高度保守的蛋白质泛素的α-β拓扑结构。我们将研究其机械设计的起源，并将其与肌联蛋白免疫球蛋白模块进行比较。通过使用蛋白质工程和诱变，我们将研究脯氨酸（以及E，V和K残基）在控制PEVK区域的弹性中所起的作用。使用各种寡聚化结构域，我们将组装成束的肌联蛋白为基础的多聚蛋白，研究肌联蛋白力学的超分子排列的效果。我们期望，除了发现新的机制，调节肌联蛋白的弹性，我们的研究将有助于进一步发展的新领域的研究单分子力谱。

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.