Single Molecule Studies of Titin Elasticity

肌联蛋白弹性的单分子研究

• 批准号: 7058755
• 负责人: Julio M Fernandez
• 金额: $ 42.68万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7058755
• 关键词: atomic force microscopy; biomechanics; chemical stability; conformation; disulfide bond; elasticity; fiber cell; immunoglobulins; molecular dynamics; muscle function; muscle proteins; proline; protein engineering; protein folding; protein isoforms; protein protein interaction; protein structure function; sarcomeres; ubiquitin

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.

描述（由申请人提供）：我们使用单分子技术来研究巨型肌肉蛋白滴定的机械设计。 Titin跨越了肌肉肌膜的一半，并且可以长在千分尺上。与肉类I带重叠的钛区域决定了肌肉弹性。 I带滴定具有特征性的模块化设计，由免疫球蛋白（IG）型域的串联重复序列组成，被富含P，E，V和K残基的区域中断，对于心脏肌肉，另一个由名为N2B的独特序列制成的区域。我们的长期目的是了解正常状态和患病状态下titin弹性的分子机制。 详细的单分子研究对组成型钛质区域的一些模块的一些模块揭示了复杂的结构依赖性机械设计。但是，通过替代其I频带的替代剪接，通过数量惊人的外显子，编码IG模块编码的106个外显子和编码PEVK序列编码的114个外显子来对TITIN弹性进行细微的调节。没有什么知之甚少，该基序由替代型的剪接区域或它们如何控制TITIN弹性所编码的机械图案。同样，已经显示出大量的TITIN IG结构域具有机械稳定二硫键的形成的潜力。此外，已经证明钛分子相互作用，表明每种弹性细丝可能由几个钛分子组成，这些分子可能形成弹性的四元结构。因此，滴定弹性可能会通过几种新型机制来细微地调节。 我们将单分子AFM和蛋白质工程技术结合在一起，以研究Titin力学的单个构件。我们将检查不同IG模块的机械特征和Titin剪接区域中的PEVK序列。我们将研究二硫键桥形成对IG域的机械性能的作用。我们将研究其他蛋白质褶皱的机械稳定性，例如肌肉蛋白乌托蛋白的螺旋线圈拓扑以及高度保守的蛋白质泛素的α-β拓扑。我们将研究其机械设计的起源，并将其与钛免疫球蛋白模块的起源进行比较。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。使用各种寡聚域，我们将组装基于钛丁物的多蛋白束，以检查上分子排列对钛机械的影响。我们预计，除了发现调节钛弹性的新机制外，我们的研究还将有助于进一步发展单分子力光谱研究的新研究领域。