Keith Weninger

DNA mismatch repair is essential in healthy cells to maintain genomic stability during genomic duplication and is involved in diseased states as its failure is directly linked to several cancers in humans as well as contributing to resistance to chemotherapy. The goal of this project is to determine the molecular interactions among repair proteins that activate mismatch repair upon detection of a DNA mismatch. These studies will reveal the basic mechanisms that underlie mismatch repair, which will be important for designing treatment of cancers involving malfunction of DNA mismatch repair.

This project aims a developing a novel analytical technique to detect the binding of proteins at the single-molecule level, in their native state without labeling, and with a time-resolution of tens of microseconds. If successful, it will provide a tool to studying assembly of protein complexes, which are pervasive in disease-relevant cellular processes.

This proposal uses a combination of atomistic simulations, single-molecule FRET experiments and statistical analysis to analyze atypical structures associated with trinucleotide repeats diseases. Given the high rate of mutation of these atypical structure, their inferences for evolution will be explored.

This project is in response to request for proposals for collaborative research addressing macromolecular interactions in cells. This project is a collaborative work between Keith Weninger at NCSU and Dorothy Erie and Eric Brustad at University of North Carolina ? Chapel Hill that is using the Multiple PI option. The focus is to develop single molecule fluorescence approaches to determine stoichiometry, temporal and spatial dynamics, and conformational changes in multiprotein repair complexes that repair DNA inside live cells. The focus will be on bacterial and yeast cells.

Our long-term goal is to understand the molecular mechanism responsible for DNA mismatch error detection and repair. In this proposal, we are focusing on the step in the mismatch repair process related to determining which strand of the newly copied genome is the original parent and which is the newly generated copy. If the mismatch repair system could not discriminate between the parent and daughter strand, it would not be clear which side of the mismatch was correct and which is an error. The parental side of a mismatch contains the original genetic code and must be used to deduce what change to make in the erroneous daughter base to effect repair. We will use single molecule fluorescence methods that can measure the dynamic motions and interactions of individual molecules in real-time to directly observe mismatch repair proteins sensing DNA errors and signaling repair. Our methods will allow the sequence of events within this process to be completely determined and will reveal the key interactions whose failure leads to the cancerous state. Because cancer is commonly caused by defects in the mismatch repair system a deeper understanding of these cellular mechanisms will allow point toward new strategies for preventing and treating cancer in humans.

Supplement for existing project: Single molecule study of the role of SNARE protein assisted membrane fusion in calcium triggered neurotransmitter release

This is a continuation of the work originally proposed. Thermionic energy conversion is typically achieved through the combination of a hot electron emitter (cathode) with a somewhat cooler collector (anode). We propose the development of thermionic energy conversion based on thermionic emission from nanostructured carbon surfaces with integrated gating and collector structures. Nanostructured carbon cathodes exhibit field enhancement factors greater than 1000, which enhances the emission current, minimizes space charge effects, allows efficient operation at temperatures less than 1000 degrees C, and relaxes the enginering constraints on emitter collector distances. The research will establish the thermionic emission characteristics of nanostructured materials developed by this team. Materials will be optimized for operation at temperatures less than 1000 degrees C. New approaches will be developed to integrate the emitter and collector materials into structures for direct conversion of thermal energy into electrical energy.

This proposal aims to develop the new capability of using single molecule fluorescence (or Förster) resonance energy transfer (smFRET) from proteins within living cells to observe real time conformational dynamics in parallel with spatial localization of individual proteins as they circulate within cellular networks. Experiments that follow single molecules can uncover properties that are impossible to observe in bulk measurements due to the inherent averaging over molecules and over time, and lack of synchronization. Single particle measurements of dynamic conformations of individual molecules can determine multiple reaction pathways and transient intermediate states as well as exact distributions of molecular properties. The general approach in this proposal is to use high resolution structural information already available for the proteins under study to design site-specific labeling mutants. These mutants will be produced, purified and labeled free of cells and then microinjected into cultured eukaryotic cells. Fluorescence microscopy and spectroscopy will be used to track individual protein molecules as well as determine the degree of FRET from that molecule in real time as they circulate within living cells. This untested approach, if successful, will have broad applicability to many biological questions and has the potential to reveal details of cellular networks that are not observable by any other technique. This approach will be applied within two different physiological networks to determine, 1) the spatial distribution and detailed sequence of folding/unfolding transitions in SNARE proteins involved in intracellular vesicle transport and membrane fusion, and 2) the activation of Protein Kinase A in second messanger signaling. Advances in understanding of cellular regulatory and signaling networks will have broad impact in all areas of health related research: understanding of the disease state, homeostasis and development. Ultimately knowledge derived with these methods will improve human health.

Proteins fold into complex shapes that are intimately linked to their function. High-resolution techniques are capable of determining static images of these structures with atomic detail, but biological function and regulation is achieved through dynamic changes in protein conformation. Single molecule fluorescence techniques have a unique capability to detect transient molecular conformations. The power of the single molecule approach arises because it avoids the averaging over molecules and over time that are inherent in ensemble measurements. Multiple reaction pathways, transient intermediate states as well as the exact distributions of molecular properties can be determined by following the time trajectories of the conformations of individual molecules, without the need for synchronization. We propose to apply single molecule fluorescence resonance energy transfer (smFRET) to make direct observation of a conformational transition in SNARE proteins that is postulated to have an auto-regulatory function. We plan a series of measurements using mutants of the proteins as well as a series of homologues of SNAREs from different species in order to gain a molecular level understanding of these transitions. SNARE proteins catalyze biological membrane fusion and an increased understanding of the mechanisms regulating their function will have widespread implications for many biological phenomena. The techniques demonstrated in this proposal will be directly applicable to investigations of conformational dynamics in other protein based macro-molecular machines.

This proposal is for the Ralph E. Powe Junior Faculty Award from Oak Ridge Associated Univeristies. The goal of this proposal is to determine whether the protein pore model or the lipid pore model is correct for Sindbis virus particles fusing with pure lipid vesicles. In the work proposed here, binding and fusion of Sindbis virus particles to protein-free vesicles will be initiated by conformational changes in the viral proteins that occur in response to low pH. Two-color fluorescent microscopy of different dyes incorporated into the membrane and into the content of the vesicles will report with high resolution the relative timing of the continuity of the interior and surfaces of the virus and vesicle. Results of these experiments will directly resolve the controversy about the composition of the fusion pore: lipid or protein.