CAREER:Experimental Studies of Protein Thermodynamics Facilitated by NMR with Reverse Micelles

Rowan University, Glassboro NJ

Investigators

Abstract

Though all cells are composed primarily of water, the environment inside the cell is quite heterogeneous, containing nanoscale spaces created by membrane-bound compartments and by high concentrations of large molecules including nucleic acids (DNA/RNA) and proteins. Generally, studies of proteins have been performed in dilute solutions because the cellular environment is too complex for most experimental approaches, thus the effects of this environment on protein structure and function remains poorly understood. This project uses nanobubbles, called reverse micelles, to mimic the cellular environment so that the influence of spatial restriction and interfacial interactions on proteins can be experimentally dissected. These experimental measurements are being compared to computer simulations of proteins inside these nanobubbles to produce a unified view of environmental effects on proteins. This approach is expected to provide a foundation for developing improved predictive models for protein function inside cells, particularly when the function of the protein is related to its structural stability. These studies are integrated with experiential education and mentorship of undergraduate students in the laboratory and the classroom including the development of a new research-based laboratory for an upper-level biophysics course aimed at helping students learn science by doing science. The project is designed to offer research experience to these students in a fashion that scales the complexity of the experiments and modeling as the students advance through their undergraduate education. In recent years, the relevance of the complex intracellular milieu in modulating protein structural stability and function has become clear. As a result, the paucity of predictive models for these effects represents an important biophysical knowledge gap. This research program combines nuclear magnetic resonance (NMR) with reverse micelle (RM) encapsulation of proteins to measure previously challenging thermodynamic environmental influences on protein structure and function. RMs are surfactant-stabilized complexes that spontaneously organize to encapsulate a nanoscale bubble of water in a nonpolar solvent. RMs have been shown to facilitate study of proteins by NMR including the ability to directly modulate their structural stability by varying the size of the bubble or the nature of the water-surfactant interface. This program pursues three specific avenues of inquiry to provide novel insight on 1. Weak electrostatic and hydrophobic forces between proteins and interfaces, 2. The magnitude of the hydrophobic effect, and 3. The effect of excluded volume on protein dynamics and disorder. Using a suite of model proteins and the tumor suppressor protein p53, which contains both highly stable folded domains and intrinsically disordered regions, these studies harness the tunability of reverse micelle systems to dissect the environmental thermodynamic driving forces of protein structure and quantify their differential contributions in unprecedented detail. These studies are integrated with an upper-division molecular biophysics undergraduate course via a research-based learning experience in which students design, execute, and report a novel study on the impact of crowding molecules on protein structural stability. The practical experience gained is reinforced through a mentorship program that matches students from underrepresented groups with STEM professionals (mentors) in their field of interest, thereby aiming to increase the percentage of underrepresented STEM graduates that successfully pursue STEM careers. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.