Intellectual merit. This project is aimed at expanding the arsenal of Electron Paramagnetic Resonance (EPR) and, especially, high resolution high field (HF) EPR methods by developing novel experimental capabilities to study fundamental roles of intermolecular interactions in self-assembly and structure-function relationships in multi component biological systems. Specific emphasis will be put on elucidating lipid-protein interactions for the Sec14 protein family. During the preceding phase of this NSF-funded project, an HF EPR-based method to separate two major components of solvent effects on spin-labeled protein residues and to detect hydrogen bond formation and local electrostatic effects was developed. While the general concepts of membrane protein folding and thermodynamic stability are beginning to emerge, the arsenal of experimental spectroscopic methods for assessing local protein electrostatics and local hydrogen bonding interactions remains severely limited. This research project aims at further developing spin-labeling HF EPR and double-resonance methods for mapping the hydrogen bonding environment for protein systems without the necessity of preparing high quality crystals. The method relies on incorporating small molecular tags, based on nitroxide radicals, into the protein structure. These labels have molecular volume and structure similar to the native protein side chain and, therefore, are known to cause only minimal perturbation to the tertiary structure. The main advantage of such labels lies in the sensitivity of their EPR spectra to the local electrostatic environment and hydrogen bond formation resulting in essential biophysical data that are difficult to obtain otherwise. The sensitivity of nitroxide spin labels to local electrostatics and hydrogen bonding is further enhanced by high field and double-resonance EPR methods under development. These methods will be further refined during this project to elucidate an intriguing and largely unknown molecular mechanism by which the Sec14 protein and its analogs regulate the interface between phospholipid metabolism and membrane trafficking. Spin label EPR and a complementary array of biophysical methods will be used to study how Sec14 recognizes, binds and transports phopholipids and how it interacts with phospholipid membranes.
Broader impact. The methods developed in the course of this project will fill the gap in the existing experimental capabilities of EPR and enable detailed biophysical studies of local electrostatics and hydrogen bonding that are directly involved in structure-function relationships of many biological systems of contemporary interest - from model membranes and peptides to ion channels, transporters and G-protein coupled receptors. The project will integrate research and teaching by adding research-driven experimental tasks to a largely lecture course "Physical Methods in Biological Chemistry" developed by the PI. In the course of this project graduate and undergraduate students will be trained across several disciplines including biophysical spectroscopy, chemical synthesis and methods of molecular biology. The project will expand research opportunities for undergraduate students, especially, minority students through the Alliances for Graduate Education and the Professoriate (AGEP) and Research Experience for Undergraduates (REU) programs. In an effort to reach out to undergraduate colleges in rural North Carolina, the PI will continue collaboration with the University of North Carolina-Pembroke. The PI and her group will be engaged in science projects and demonstrations in the Centennial Middle School associated with NCSU that will be coordinated through the Science House, a North Carolina institution for K-12 outreach program.
