Our research broadly aims at understanding the mechanism of action of enzymes. We use a combination of pre-steady state kinetics, single molecule methods, structural and biophysical approaches to build quantitative models of enzyme activity to understand how they function in the cell. Currently, we are focused on enzymes that function in two distinct biological phenomena: DNA Repair & Recombination and Electron Transfer
DNA Repair & Recombination Genomic stability is maintained by the concerted action of multiple DNA repair pathways in the cells. Mutations that lead to defective DNA repair are hallmarks of cancers and their underlying pathologies. Dozens of enzymes orchestrate specific DNA repair and recombination processes and we are exploring how these enzymes recognize damages in the DNA and coordinate with the cell cycle and replication machinery to correct these damages. Pokhrel et. al. NSMB. 2019 Yates et. al. Nat. Comm. 2018 Pokhrel et. al. NAR 2017. Lytle et. al. JMB 2014.
Oxidoreductases and Electron Transfer We are exploring how two oxidoreductases, Nitrogenase and DPOR, use ATP to drive substrate reduction. Nitrogenase catalyzes the reduction of dinitrogen to ammonia and uses 16 ATP molecules per substrate reduced. The dark operative protochlorophyllide oxidoreductase (DPOR) complex similarly reduces Pchlide to Chlide and substrate reduction is coupled to ATP hydrolysis. Both these multi-protein assemblies orchestrate step-wise molecular events to enact substrate reduction. These steps are associated with large conformational changes in the protein complex. Our long-term goal is to understand how ATP is utilized to orchestrate substrate reduction. Danyal et. al. PNAS 2016. Duval et. al. PNAS 2013.
Non-canonical Amino Acids and Fluorescent Proteins Multiple proteins often work together to complete a biological process. For example, during DNA repair, more than a dozen enzymes work on a single DNA template. Experimental tools to follow a single enzyme in this multi-protein milieu are required to elucidate their mechanism of action. We are utilizing non-canonical amino acids (ncAA) to generate fluorescent versions of our proteins of interest such that they produce a change in fluorescence upon binding to DNA. Exploiting this approach, we are currently investigating the assembly of enzymes during Homologous Recombination, Nucleotide Excision Repair and Base Excision Repair. Pokhrel et. al. NAR 2017.
Join Our Team We welcome motivated postdoctoral scholars, graduate & undergraduate students to join our team. We also welcome middle and high school students seeking to perform research in the summers. Our contact information can be found here: CONTACT
Our research is supported by grants from the following federal and private agencies