To thrive, cells must regulate dense networks of protein-protein interactions that drive basic processes such as cell growth, division, and death. One well-known regulatory mechanism is "post-translational phosphorylation" - the reversible addition of phosphate groups to specific amino acids within a protein. Phosphorylation can alter protein binding specificity, enzymatic activity, or sub-cellular location, thereby regulating its interaction with other proteins. But just how this comes about is not yet clear. In fact, an atomic level explanation for how site-specific phosphorylation changes a protein's interaction properties remains an open challenge. This project addresses this challenge by investigating the effects of post-translational phosphorylation on protein conformational dynamics. The protein segments subject to phosphorylation are often the same segments involved in intermolecular interactions. Critically, they often have conformational flexibility that defies explanation by standard biochemical analysis. By investigating how these dynamic protein segments convert phosphorylation into functional change, this project will provide insight into a widespread, yet poorly understood aspect of protein phosphorylation. Gaining this insight is a necessary step toward understanding and manipulating protein interaction networks. This project will bring interdisciplinary educational opportunities to students at early stages of college by developing freshmen courses that introduce physical principles based on biophysical phenomena. This project will also train doctoral students and undergraduate research interns, including members of underrepresented groups, to become highly qualified scientists in the area biophysics and computational biology; the cultivation of such expertise is vital to keep the U.S. competitiveness in research and in the global economy.
The project will combine liquid-state multi-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, molecular dynamics (MD) simulations, and mutagenesis to map how site-specific phosphorylation impacts protein dynamics and protein-substrate interactions in the mitotic signaling protein, Pin1. Pin1 interactions with other proteins are sensitive to both conformational dynamics and post-translational phosphorylation. This proposal will determine the extent to which phosphorylation exploits electrostatic networks to achieve long-range site-to-site communication (allostery). This project will also define how phosphorylation of intrinsically disordered regions nevertheless elicit functional changes at remote binding sites. Because Pin1 interacts with numerous other signaling proteins, results of this project will give broad insight into the mechanisms by which dynamic protein segments convert phosphorylation into functional change. This project will also include development of general protein NMR methods to better characterize dynamic electrostatic interactions in proteins, and their description in terms of conformational ensembles. This project is supported by Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.
