Structural investigations of DNA oligonucleotides and the peptide hormone calcitonin in the solution phase via NMR spectroscopy and molecular dynamics

Edward Phillip Nikonowicz, Purdue University

Abstract

Assignment of the $\sp1$H and $\sp{31}$P NMR spectra of two oligonucleotide duplexes, an extra-helical adenosine tridecamer d(CGCAGATATCGCG)$\sb2$, and a tandem G $\cdot$ A mismatched base pair d(CCAAGATTGG)$\sb2$, has been made by two-dimensional $\sp1$H-$\sp1$H and heteronuclear $\sp{31}$P-$\sp1$H correlated spectroscopy. J$\sb{H3\sp\prime-P}$ coupling constants for each of the phosphates of the tridecamer and decamer duplexes was obtained from the $\sp1$H-$\sp{31}$P J-resolved selective proton flip 2D spectrum. By using a modified Karplus relationship the C4$\sp\prime$-C3$\sp\prime$-O3$\sp\prime$-P torsional angles ($\epsilon$) were obtained. There exists a good linear correlation between $\sp{31}$P chemical shifts and the $\epsilon$ torsional angles for both oligonucleotides. A complete relaxation matrix approach employing a matrix eigenvalue/eigenvector solution to the Bloch equations avoids the approximation of the two-spin method. The structures of the extra-helical adenosine tridecamer and a tandemly positioned G $\cdot$ A double mismatch decamer oligodeoxyribonucleotide duplexes have been calculated by an iterative refinement approach using a hybrid relaxation matrix method combined with restrained molecular dynamics calculations. Distances from the 2D NOESY spectra were calculated from the relaxation rate matrix which has been evaluated from a hybrid NOESY volume matrix comprising elements from the experiment and those calculated from an initial structure. Although the crystal structure of the tridecamer clearly shows the extra-helical adenosine looped out away from the duplex, the NOESY-distance restrained hybrid matrix/molecular dynamics structural refinement establishes that the extra-helical adenosine stacks into the duplex. Starting from both A- and B- DNA and mismatch A syn and A anti models, structures for the decamer have been calculated which are in good atomic RMS agreement with each other ($<$1.6 A rms) but which differ from the reported crystal structure ($>$3.6 A). Further refinement of the decamer in a box of TIP3P water with counterions indicates that the "bent" structures derived in vacuo are energetically as favorable as "straight" model-built and crystal structures. Finally, the application of $\sp1$H NMR methods to the solution structure of the peptide hormone Salmon calcitonin I has shown that this molecule adopts an alpha-helical state for a portion of the sequence in methanolic solution. (Abstract shortened with permission of author.)

Degree

Ph.D.

Advisors

Gorenstein, Purdue University.

Subject Area

Biochemistry

COinS