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.
Understanding the forces that govern the interaction of proteins with one another assists in the understanding of such processes as macromolecular assembly, chaperone-assisted protein folding and protein translocation. We study the polymerization of sickle cell hemoglobin as a paradigm for understanding protein-protein interactions. Polymerization of sickle cell hemoglobin results from the one residue mutation (b6 Glu to Val) in the A helix of the protein. This one residue mutation creates a hydrophobic surface that initiates the aggregation of the protein tetramers, by interacting with the b85 Phe and b88 Leu residues on an adjacent tetramer. Our studies are designed to investigate the polymers as they are forming by monitoring the Phe Raman bands, which are reflective of local environment. At present we have established that the tertiary structure of Hb S tetramers differs from that of Hb A in the region of the mutation. This tertiary structural change may have implications for polymer formation. Our current work focuses on a Hb S derivative, which allows us to chemically induce polymerization of the molecule. The mechanism of polymerization is the same for the modified Hb S and native Hb S, as shown by kinetic measurements. Electron micrographs of polymerized Hb demonstrate that the fibers from the modified Hb S are similar in size and shape to those formed by deoxy Hb S. Further work includes a study of Hb S fibers at different stages in the polymerization process and a study of the effect of anti-sickling drugs on Hb S fibers and tetramers.
Funding: National Science Foundation
Current lab members: Andrew Moreno, Yan Li, Jacob Litke, Rachel Cohen, Hyo Yang, Mahmoud Saleh, Anwesha Bhattacharya.