Single Molecule Approach to Biological Physics
We study several different problems in molecular biophysics using the common methodology of single molecule fluorescence. This means what it sounds like – we measure behavior of one molecule at a time. This is very powerful for systems where all molecules are not behaving exactly the same or can’t be synchronized in time.
DNA mismatch repair
Our studies are aimed at revealing the molecular-level mechanisms used by the cellular protein system that locates base mismatch errors and some types of damage in DNA and then signals for their repair. Defects of this protein system are involved in multiple types of cancer including colon (especially hereditary non-polyposis colorectal cancer), ovarian, prostate, bladder and sporadic leukemia. Mutations in these cellular proteins that can occur during the course of tumor growth often result in development of tumor resistance to chemotherapeutic treatments.
We engineer DNA substrates and recombinant versions of the mismatch repair proteins to allow attachment of fluorescent dyes at specific locations. We then use the FRET method to record realtime information about how the conformations of these molecules change as they encounter mismatches and recruit partner proteins that carry out repair.
Our project is a collaboration with Dorothy Erie’s group at UNC-Chapel Hill
We use single molecule FRET to characterize the protein complexes formed from SNARE proteins and other regulatory proteins that are involved in neurotransmitter release.
We study membrane fusion involving SNARE proteins as well as enveloped viruses. The general approach involves fluorescent dyes in the interior or in the membrane of viruses and synaptic vesicles. When they fuse with a target membrane like a supported lipid bilayer, then the dyes mix and we observe the dynamic process.
Protein Conformational Dynamics
We used single molecule FRET to investigate conformational dynamics of several different proteins. Most recently we have been focused in a class of proteins known as intrinsically disordered. These proteins don’t fold into a single conformation, but rather dynamically exist in an ensemble of random’ish’ configurations. The details of these behaviors and transistions in these behaviors upon interacting with signalling partners is a key unknown that we are pursuing. These experiments have focused on neuronal proteins and also on a family of proteins that appear to play a role in prostate cancer.
Some of this work is a collaboration with Mark Bowen’s group at Stony Brook
We are always interested in pushing technology. We work toward increasing temporal resolution, number of simultaneous colors in single molecule FRET, and using polarization or other optical properties to gain additional insight into our samples.
Our project is a collaboration with Robert Riehn’s group here at NCSU.