Protein and nucleic acid structure; structure-function relationships in protein-nucleic acid and protein-protein interactions using UV resonance Raman spectroscopy
We employ fluorescence and UV resonance Raman spectroscopic methods for probing protein-nucleic acid and protein-protein interactions. The Raman effect can be enhanced by several orders of magnitude by exciting into or near to an absorption band. Thus, the excitation wavelength can be used to probe different regions of the macromolecules. For example, an excitation wavelength of 230 nm selectively investigates the aromatic residues, Tyr and Trp; whereas, 260 nm selectively probes nucleic acid residues. We exploit the resonance effect to separately investigate DNA conformation from protein structure.
Protein-Nucleic Acid Interactions
Nucleotide-binding proteins play an extremely important role as regulators of genomic function. However, the molecular mechanism of these processes is not well understood, since only a few crystal structures exist for protein-nucleic acid complexes. We are addressing the mechanism of protein-mediated regulation of genetic processes such as repression, recombination or expression by investigating the nucleotide-protein interface for a class of prokaryotic his tone-like proteins. The stabilization of DNA in coil or loop structures is the postulated mechanism by which these proteins participate in replication and inversion reactions and also enhance binding of proteins such as Lac repressor and camp-activator protein. It is this protein-induced deformation of DNA structure, which in turn modulates its genetic function, that motivates our investigations.
We are studying the HU and IHF proteins from E.coli, which bind to the minor groove of DNA through two flexible β-strand regions. This type of interaction is of interest since the majority of previously characterized protein-nucleic acid interactions have typically involved direct contact between the protein a-helix and the major groove. The sequence specificity of the minor groove interaction is examined by monitoring H-bond pairings of nucleotide exocyclic amino and carbonyl groups. The vibrational modes of these exocyclic groups reflect the formation of H-bonds since molecular vibrations are dependent on the masses of the vibrating atoms, the molecular geometry, and the forces that restrain molecules in their equilibrium positions. We are also using fluorescence spectroscopy to probe the binding interaction to gain information regarding the global conformation of the protein-DNA complex. Our experiments focus on utilizing either the natural fluorophores in the protein (e.g. Tyr or Trp residues) or labeling the protein or the DNA with a fluorescent molecule. These fluorescence measurements allow us to probe the conformation of the DNA before and after it binds to the protein and fluorescence resonance energy transfer measurements reveal the relative proximity of the protein to the DNA.
Current lab members: Zane Lombardo, Dacheng Zhao, Charya Khun, Nick Taylor, Meera Joshi