Structure elucidation of DNA decamers, decamer-drug complexes and a human growth hormone peptide by nuclear magnetic resonance spectroscopy

Robert Powers, Purdue University

Abstract

The solution structure of the DNA decamers, the (CCCGATCGGG)$\sb2$- (N-Me CYS 3, N-Me CYS 7) TANDEM and (CGCTTAAGCG)$\sb2$-(+)-CPI-CDPI$\sb2$ DNA-drug complexes and the 28 amino acid N-terminus fragment of the human growth hormone peptide were analyzed primarily by $\sp1$H and $\sp{31}$P nuclear magnetic resonance. The solution structures were modeled by using either AMBER with "two-spin" distances or an AMBER/MORASS protocol using NOE volumes. Experimental results obtained from UV melting curves, imino melting profiles and $\sp{31}$P NMR data suggest that the decamer, (CCCGATCGGG)$\sb2$ exists in multiple states and conformations, including a possible hairpin loop. While (N-Me CYS 3, N-Me CYS 7) TANDEM was expected to bind to decamer by bis-intercalation, the results were consistent with a non-intercalation mode of association with points of contact between the second and fifth phosphate from the ends. Modeling and molecular mechanics energy minimization demonstrated that novel major groove and minor groove structures are possible. In addition, the minor groove complex suggests a possible pre-bis-intercalation model. The (+)-CPI-CDPI$\sb2$ analog was determined to be the best choice for a detailed NMR study based on the UV analysis of the CC-1065 analogs bindings to polymer DNAs. The presence of (+)-CPI-CDPI$\sb2$ induced an asymmetry in the NMR spectra and 1.0 ppm upfield shifts in the $\sp{31}$P spectra and 0.5 ppm upfield shifts in the $\sp1$H NMR spectra. The effect on the $\sp{31}$P spectra was time dependent and inferred a binding heterogeneity. This was supported by additional resonances in the $\sp1$H NMR spectra which could not be explained by one configuration. (+)-CPI-CDPI$\sb2$ was found to bind in the decamer's minor groove and alkylate the N3 of A17 based on the observed effects of the decamer's chemical shifts. A relationship was found between the decamer's $\sp{31}$P chemical shifts and $\sp{31}$P-H3$\sp\prime$ constants. This implied a direct relationship between $\sp{31}$P chemical shifts and the local P-O backbone torsion angles. The $\sp{31}$P chemical shifts and the $\sp{31}$P-H3$\sp\prime$ coupling constant were found to correlate with the model built structures' epsilon torsional angles. The structure's helical twists values also corresponded to helical twists values calculated from Calladine's rules. The 28 residue N-terminus fragment of the human growth hormone was shown to have considerable $\alpha$-helical content in water at pH 3.0. The $\sp1$H NMR and circular dichroism indicate that residues 8 to 24 correspond to the $\alpha$-helical region. This was supported by the ability to model the peptide as an amphiphilic helix. The CD and gel filtration results support a dimer formation of the HGH peptide.

Degree

Ph.D.

Advisors

Gorenstein, Purdue University.

Subject Area

Biochemistry

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