Quantitative Studies of Intrinsically Disordered Protein Structure and Function

In this research project broad principles wil be developed to describe the structure of highly flexible proteins, which will provide predictive insight into their function. The PI's new models connecting molecular structure and function for highly flexible proteins will lead to the routine quantitative characterization of these molecular systems. A new experimental methodology will be implemented for atomic resolution characterization of the structures possessed by highly flexible proteins and the changes to those structures that often accompany binding to other macromolecules. As a result, this research and associated training activities will yield fundamental, molecular level insights into essential biological processes that will benefit the biotechnology industry. This program will train junior scientists to seek fundamental insight into the systems and processes from biochemistry, using the principles and quantitative laws from the physical sciences. During each year of the project, the PI will bring middle school students from disadvantaged backgrounds to Penn State University in order to illustrate for them the opportunities that arise from higher education in science and engineering fields. Additionally, the PI has been named a mentor for two Penn State programs that aim to bring under-represented minority undergraduate students into the research laboratory. Through these programs, the PI will bring a minimum of one enrolled student per year of the project into his laboratory. Trainees supported by this project will continue to provide the daily mentorship of all students engaging in these outreach programs, with guidance from the PI, who is firmly committed to expanding opportunities in science for students from diverse backgrounds.

The research objective of this project is to quantitatively describe intrinsically disordered protein (IDP) ensembles and structure-function relationships on the atomic and molecular scale. The challenge is to find a model that best describes the conformational features of highly flexible biomolecules, as this structural description is intimately connected with cellular function. From a broader point of view, the objective is to develop new models for protein conformation and dynamics that lead to the application of structural biological tools to IDPs, which are hypothesized to possess native structure that is directly responsible for imparting their specific functions. Defining structure-function relationships for IDPs requires broadening the traditionally narrow paradigm developed for cooperatively folding systems. This project continues efforts to develop quantitative and efficient tools for experimentally constraining IDP structure and site-specific interactions with other biomolecules through carbon-detected NMR spectroscopy. Traditionally acquired (i.e., 1H-detected) NMR spectra of IDPs suffer from poor chemical shift dispersion, rendering them unsuitable to high resolution applications in most cases. It has been shown that 13C-direct detection spectroscopy generally produces spectra suitable for quantitative analysis of IDPs. The development of carbon-detected NMR methods for the study of IDPs will continue to be developed during the course of this project. Recently a fully atomistic structural ensemble of the protein FCP1 in its unbound state has been resolved. This project will expand efforts to include structural characterization of the complex formed between FCP1 and its folded partner Rap74, while also defining the conformational ensemble of a segment from the RNA Polymerase II C-terminal domain, which is the biological target of the FCP1 phosphatase activity. Finally, it is necessary to connect structural findings to functional outcomes. For the model systems investigated in this project, biological function can be assayed by defining binding interactions between the IDPs under study and their cooperatively folded partners. Functional hypothesis testing will be primarily carried out through quantitative calorimetric binding studies, establishing the energetic consequences of intrinsic disorder and defining the driving forces for disordered protein interactions.