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Victor Hsu
Associate Professor, Biochemistry and Biophysics
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RESEARCH:
Our laboratory is interested in the application of nuclear magnetic resonance (NMR) spectroscopy to study the structural aspects of biomolecular interactions, especially in protein-nucleic acid complexes. These interactions account for many of the major cell functions such as the induction and/or repression of gene expression and the packaging of nucleic acids into other superstructures. NMR techniques are uniquely suited for studying biomolecular structures at atomic resolution as well as their dynamic behavior in solution.
We are studying both sequence-specific and nonspecific DNA-binding proteins and their complexes with synthetic DNAs. Recent advances in NMR spectrometers and multidimensional experiments, coupled with the current molecular biology methodologies for producing large quantities of isotopically labeled (primarily 2H, 13C, and 15N) proteins, have made it possible to study protein-DNA complexes and to elucidate the details of their interactions. An intriguing question that we are also investigating is how changes associated with aging and cancer effect the structures and interactions of proteins and DNA.
One protein that we have been studying, the Transcription Factor 1 (TF1) from Bacillus subtilis, not only binds to specific DNA sequences, but also shows a marked preference for binding sites which contain 5-hydroxymethyluracil (hmU) bases. While hmU occurs naturally in some bacteriophages, it has also been identified as a mutagenic lesion resulting from oxidative attack on thymine in normal DNA. We have been active in developing protein NMR strategies involving specific deuteration of selected amino acids, 15N-labeling and three-dimensional experiments to overcome resonance overlap, assignment ambiguities, to obtain more accurate interproton distances for use in subsequent structure calculations, and to investigate the intrinsic dynamics of the protein backbone.
We are investigating whether hmU recognition by TF1 is due to any conformational changes in DNA induced by hmU incorporation. To this end we have synthesized hmU-containing oligonucleotides as well as their thymine-containing analogs and are proceeding with determining the detailed structures of the DNA oligomers using a combination of proton, carbon, and phosphorus NMR experiments. In particular, since the NMR-derived distance constraints that can be used in DNA structure calculations are somewhat limited in their usefulness, we have been working on correlating heteronuclear NMR coupling constants with torsion angles to better define the solution conformation of the oligonucleotides.
We are also interested in studying other DNA-binding proteins and their mutants to provide insights into what factors most affect protein stability and how DNA-binding is mediated. We plan to use isotope-edited NMR techniques to specifically observe intra - and/or intermolecular interactions.