The research project aims to utilize an electron paramagnetic resonance (EPR) based methodology to study the structure and conformation of a class of proteins that are termed natively unstructured, meaning they have no stable three dimensional structures. Nearly one third of proteins are predicted to function as unstructured motifs, and very few biophysical methods can be utilized to investigate this class of proteins. Through this research, the EPR based methods will be developed, and two classes of proteins will be investigated. The first is a peptide-based inhibitor of an aspartic protease. Results from these studies can be important for understanding how natural peptides are utilized as enzymatic inhibitors, thus potentially providing information about the rational design of new classes of chemicals or peptide-based inhibitors. The second protein is involved in regulating many cellular pathways. Results from this study will provide insights on how proteins can regulate multiple pathways without adopting a well defined structural motif.
The participation of undergraduate students (particularly women and those from underrepresented groups) in research in biological magnetic resonance, which spans the fields of biology, chemistry and physics, serves as the central theme of this CAREER project that links research and educational objectives. Specifically, this integration is obtained by a three-tiered approach. One aspect is the recruiting of students into the research laboratory from undergraduate biochemistry and biophysical chemistry labs, where students can directly apply the theory and experimental skills learned in the classroom to a real research setting. The second aspect is the targeting undergraduate students within their first year of college. At The University of Florida (UF), a variety of programs are already in place that provide pools of students for this purpose: underrepresented minorities from the University Minorities Mentoring Program (UMMP), UF undergraduate students (Beckman Scholars, HHMI, REU), and high-school students from the Student Science Training Program (SSTP), organized by UF's Center for Pre-collegiate Education and Training (CPET). The third tier is the development of a summer research exchange program, with other EPR groups within the Southeastern USA, that aims to provide cross-discipline training for undergraduates in both physics and biology as related to EPR spectroscopy.
This project is receiving co-funding from the Experimental Physical Chemistry Program of the Chemistry Division.
Intellectual Merit:The major goals achieved during this funding period were the development and application of site-directed spin-labeling (SDSL) EPR methods for analysis of highly dynamic systems such as intrinsically disordered proteins (IDPs) and conformational sampling of dimeric enzymes. To date, 17 manuscripts, which have been published or are in press (2 are invited review articles) with 4 more under revision or in preparation, have resulted from the support of the grant. The major accomplishments during the funding period include: (i) demonstrating how various spin-labels, line shape parameters and high-frequency EPR can be used to assess helical structure and conformational transitions in IDPs; (ii) demonstrating that the pulsed EPR methodology referred to as double electron-electron resonance (DEER) can be used to characterize protein conformational sampling; specifically applied to HIV-1 protease (PR), a dimeric enzyme; (iii) developing data analysis and error evaluation schemes for pulsed EPR data to determine ligand induced shifts in the conformational sampling profiles; and (iv) showing how amino acid substitutions can alter the fractional occupancy of the conformational ensemble of HIV-1 PR, where some of these substitutions are distal from the active site and modulate enzymatic activity and inhibition. In addition solution NMR spectroscopy was utilized to characterize backbone dynamics; thus making correlations between dynamics and conformational sampling. The effects of co-solutes and methods of freezing in pulsed EPR data collection on results for enzyme conformational sampling were also explored. NMR data were again used to corroborate ligand induced shifts in conformational sampling observed with DEER at frozen temperatures related to the time scale of dynamics of interaction between the protein-inhibitor at physiological temperatures. Broader Impact: Funds from this NSF funded project were leveraged where two graduate students successfully obtained American Heart Association predoctoral fellowships; the fellowships only paid for their salaries, not tuition or research expenditures. These successful achievements facilitated research productivity and increased the number of students (graduate and undergraduate) that could be supported by this grant. Funds from this grant directly supported 5 high school students, 13 undergraduates, 9 graduate students; with 7 having obtained their PhDs and three obtaining MS degrees. Undergraduate students, both female and those from underrepresented groups, were recruited from undergraduate lab courses (Biochemistry and Biophysical chemistry) that I taught (9). Others (8 to date) were recruited as freshman into the research laboratory through participation in UF’s HHMI Science for Life (S4L), Minority Mentoring (MMP), and Beckman Scholars programs. Together, these students often co-authored publications (some first author) that resulted from their four years of work. Funds to support research were also utilized in the training and development of high-school students through participation in UF’s Student Science Training Program (www.cpet.ufl.edu/sstp/SSTPContact.html). This is a highly structured 7 week research immersion experience. High-school students were paired with a graduate or undergraduate mentor. They also attend group meetings and the summer course I taught in Biochemistry Lab methods in recombinant protein expression, purification and enzymatic characterization – skills they directly utilized in achieving the goals of the research of this project. Funds from this proposal supported undergraduate and graduate student exchange experiences with collaborators as well as their travel to workshops and to meetings.
