NMR Studies of Protein-DNA Interactions: Determinations of DNA Structures Recognized by Bin Recombinase and Studies of Their Roles in Protein Binding Interactions

Citation

Sun, Yun (1992) NMR Studies of Protein-DNA Interactions: Determinations of DNA Structures Recognized by Bin Recombinase and Studies of Their Roles in Protein Binding Interactions. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/em1y-p761. https://resolver.caltech.edu/CaltechTHESIS:09132011-160134197

Abstract

The solution structures of two DNA oligomers were determined by the 2-D NMR method. These 14-base-pair DNA molecules contain the recognition sites for Hin recombinase. In spite of the differences in their sequences, the two structures are remarkably similar. The refined DNA structures possess a significant bend (25-32°) in the middle of the helices. As a result of the bending, the nearby major groove is compressed at almost exactly the position where the recombinase binds. The DNA molecules were also found to have a deepened and narrowed minor groove near the continuous dA tracts, where the minor groove contact happens between the N-terminal residues of the recombinase binding domain and the DNA molecules. Such pre-existing unique features of the free DNA molecules are likely to contribute to the specific interaction of the protein and the DNA tracts. Structure determinations by the NMR method were preceded with the use of complete relaxation matrix analysis and restrained molecular dynamics. A data processing system were developed which allowed us to simulate the NMR spectra and quantifying intensities from an overlapped data set. A complete system for high-resolution structure determinations in solution were set up and evaluated.

The conformation of the Hin 52mer peptide-the binding domain of the Hin recombinase-and its binding interactions with the DNA oligomers are studied by NMR, circular dichroism and chromatographic methods. The conclusion is that the peptide does not have a unique and stable conformation alone as a single monomer in solution. The Hin peptide can be prevented from being aggregated by adjusting to acidic conditions, and it can be folded to a stable tertiary structure in an artificial environment with small amounts of trifluoroethanol. The Hin 52mer peptide conformation is greatly stabilized or induced by the presence of the DNA bearing specific binding sequences. The DNA binding activities of the peptide may be assayed by a chromatographic method. The behavior of the peptide in the binding complex and the characteristic structural features of the DNA molecules suggest the active role of the DNA in protein-DNA interactions providing complementary interactions with the peptide and stabilizing the peptide conformation upon its binding.

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