NSF/MCB BSF: Direct Force measurements and analysis of intrinsically disordered proteins

生物体内的许多重要结构都是由蛋白质组成的，蛋白质是一种大的线状分子，通常会卷曲（“折叠”）成一个明确的球形。然而，最近的发现发现，有相当大的一部分蛋白质（包括人类中约30%的蛋白质）不会折叠;相反，它们的形状像蠕动的意大利面条一样不断波动。这些无序蛋白质也通过其组成与折叠蛋白质区分开，并且与折叠蛋白质相比通常含有更多数量的带电亚基。该项目将研究高电荷组成与无序蛋白质的波动结构之间的联系。使用现代物理方法进行的实验，将允许对蛋白质形状及其波动的新见解。该项目将特别关注在肌肉和神经元中发现的重要蛋白质。该研究项目将对培养年轻科学家以及形成国际合作产生广泛而长期的影响。内在无序蛋白（IDP）通常富含带电残基，并通过磷酸化进行调控。因此，静电相互作用在定义和调节链结构中占主导地位。对于IDP，结构是一个动态的概念，因为蛋白质在大量可用构象a中波动。该提案的总体目标是研究静电相互作用对动态IDP结构的影响。该项目将专注于两个重要的无序蛋白质：i）肌肉蛋白titin的PEVK区域，这是一个高电荷结构域，其熵弹性有助于定义肌肉的被动弹性; ii）神经肌肉中间丝（NIF）蛋白的C末端尾部，其静电相互作用驱动细丝组装成水凝胶网络，定义轴突的细胞骨架。PEVK和NIFs的动态结构将通过使用定量实验方法和软物质物理学的概念进行研究，包括低力单分子拉伸，以从熵弹性和小角X射线散射（SAXS）测量中推断动态结构。将通过探索不同溶液盐条件对结构的影响，以及探索磷酸化和不同链电荷分布的影响来研究静电效应。SAXS和单分子操作测量的组合将导致定量，微观洞察生物相关的静电控制IDP结构的机制。 这个美国/以色列合作项目得到了美国国家科学基金会和以色列两国科学基金会的支持。

Many important structures within organisms are built out of proteins, large string-shaped molecules that typically coil up ('fold') into a well-defined globular shape. However, recent discoveries have found that there is a rather large subset of proteins (including about 30% of the proteins in humans) that do not fold; instead, their shape continually fluctuates like a wriggling piece of spaghetti. These disordered proteins are also distinguished from folded proteins by their composition and typically contain a larger number of electrically-charged subunits in comparison to folded proteins. This project will investigate the link between the highly-charged composition and the fluctuating structure of disordered proteins. The experiments conducted using modern physical methods that will permit new insight into protein shape and its fluctuations. This project will particularly focus on important proteins found in muscle and in neurons. This research project will have broad, long-term impacts in training young scientists, as well as forming an international collaboration. Intrinsically-disordered proteins (IDPs) are frequently enriched in charged residues, and subject to regulatory control through phosphorylation. Thus, electrostatic interactions are dominant in defining and modulating chain structure. For IDPs, structure is a dynamic concept, as the proteins fluctuate through a large ensemble of available conformationa. The overarching goal of this proposal is to study the effects of electrostatic interactions on dynamic IDP structure. This project will focus on two important disordered proteins: i) the PEVK region of the muscle protein titin, a highly-charged domain whose entropic elasticity helps define the passive elasticity of muscle; and ii) the C-terminal tails of neuromuscular intermediate filament (NIF) proteins, whose electrostatic interactions drive the assembly of filaments into hydrogel networks that define the cytoskeleton of axons. The dynamic structure of PEVK and NIFs will be studied by using quantitative experimental methods and concepts derived from soft-matter physics, including low-force single-molecule stretching to infer dynamic structure from entropic elasticity, and small-angle X-ray scattering (SAXS) measurements. Electrostatic effects will be studied by exploring the effect on structure of different solution salt conditions, as well as exploring the effects of phosphorylation, and of varying chain charge profiles. The combination of SAXS and single-molecule manipulation measurements will lead to quantitative, microscopic insight into biologically-relevant mechanisms of electrostatic control of IDP structure. This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.