National Center for
Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, CZ-611 37 Brno, Czech
Republic
Short DNA hairpins play a significant role in a number of
biological processes. The most
interesting feature of the
oligonucleotides with a general sequence d(GCGNAGC) (N = A, G, C, T) is their
extraordinary stability represented by high melting temperatures,
polyacrylamide gel mobility and resistance against nucleases. Detailed
knowledge of these structures helps understand their unique behavior.
The d(GCGAAGC) structure has been solved previously by NMR spectroscopy
using the NOE-derived distances, torsion angles and residual dipolar couplings
[1]. The aim of the present study is a new structure calculation based on NOE
data measured at temperatures below 273 K when the intramolecular motions and
the chemical exchange of the amino protons are inhibited. Compared to the
studies at the ambient temperature, this approach allows us to extract more
distance restraints for the use in the molecular dynamics calculations.
A series of
the 2D-NOESY spectra with the mixing times in a range of 50 to 400 ms has been
measured at 268K. The assignment of the spectra revealed 11 of 14 assigned
amino proton resonances compared to 6 of 14 resonances extracted at 298K. The
NOESY cross-peaks for the different mixing times were integrated using the
SPARKY program and the results were employed to derive the inter-proton
distance restraints. Our treatment of the NOESY cross-peak volumes includes
both the Isolated Spin Pair Approach (ISPA) and the relaxation matrix approach represented
by the MARDIGRAS and MORASS algorithms in order to account for the spin
diffusion. The choice of the method for the extraction of a sufficiently large
number of accurate proton distances from 2D NOE cross-peak intensities may be important for the subsequent
structure calculations. Here we compare the NOE-derived interproton distances
obtained by the ISPA and the relaxation matrix analyses for several sets of
experimental data.
1. P. Padrta, R. Štefl, L. Králík, L. Žídek, V. Sklenář, J. Biomol. NMR,
24, (2002), 1 - 14
2. U. Schmitz, D. A. Pearlman, T. L. James,
J. Mol. Biol., 221, (1991),
271 – 292