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

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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.