Probing Reaction Pathways in Protein-DNA Complexes by Two-Dimensional Fluorescence Spectroscopy and Single-Molecule Correlation Spectroscopy

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

This award in the Chemistry of Life Processes (CLP) program supports work by Professor Andrew H. Marcus at the University of Oregon to carry out fundamental studies to investigate the local base conformations and conformational dynamics at pre-selected sites within functional DNA-protein replication complexes and to determine key structural end-states, intermediates, and transition states along enzymatic reaction pathways of these complexes. These experiments employ DNA constructs containing two adjacently positioned fluorescent nucleic acid base analogue probes, which absorb in a spectral region where DNA and proteins are transparent. Coupling between the electronic states of the dimer probe residues produces signature spectral features, which are used to infer local nucleic acid base conformation and time-dependent local motions, which is information central to understanding the mechanisms by which these complexes carry out their specialized functions. These studies will be carried out using two-dimensional fluorescence spectroscopy (2D FS, a fluorescence-detected version of 2D electronic coherence spectroscopy) to elucidate analogue dinucleotide probe conformation, and single-molecule Forster resonance energy transfer (smFRET) and fluorescence-detected linear dichroism (smFLD) to monitor conformational dynamics on time scales ranging between tens-of-microseconds to tens-of-seconds. The work supported will add to our basic understanding of the dynamic processes whereby the macromolecular machines of gene regulation and expression perform their biological functions. Potential benefits range from an improved understanding of the molecular level interface between chemical and biological behavior, to new general insights about the underlying principles of nano-machines. A significant component of the research and educational activities also involves mentoring the next generation of scientists, and encouraging the participation of students from under-represented groups. These activities include i) the involvement of undergraduate students in research projects, and providing them with guidance to develop professional careers; ii) the development of new courses at the undergraduate and graduate levels within the UO Chemistry Department; and iii) outreach to local high school students through the creation of advanced placement chemistry courses that are taught on furlough days. Graduate and postdoctoral students working on these projects will benefit from cross-disciplinary training in physical chemistry, biological sciences, and theory, enhancing their future career opportunities.