NMR DISTANCE RESTRAINTS AT LOW TEMPERATURES:
AN APPLICATION TO DNA HAIRPIN
Michaela Matějková, Petr Novák, Radovan Fiala, Petr Padrta and Vladimír
Sklenář
National Center for
Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2,
CZ-611 37 Brno, Czech Republic
Number of the
experimental NMR distance restraints extracted at the ambient temperature is
remarkably decreased by an internal mobility in a molecule, mostly chemical
exchange. This problem can be avoided by NMR measurements at lower
temperatures. To achieve the lowest temperature while keeping the liquid state
and minimize the chemical exchange, the NMR experiments can be performed using
the slowly cooled capillaries [1].
The aim of the present study is to
investigate possibilities of NMR measurements in liquid state at the
temperatures below 273 K and applicability of this approach to a nucleic acid
fragment. For this purpose, the d(GCGAAGC) hairpin has been chosen because its
structure has been previously solved precisely by NMR spectroscopy [2]. We have
performed a new structure calculation of this molecule using the low
temperature NMR measurements and compared the precision of the newly calculated
and known structures.
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
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. Then, the numbers of distance restraints extracted at 268
K and 303 K were compared. In case of nonexchangeable protons, these numbers
were comparable due to the significant spectral overlaps in the NOESY spectra
measured at 268 K. While, there was almost twice higher number of exchangeable
proton distances extracted at 268 K compared to 303 K and these restraints
helped increase the precision of the calculated structure of the d(GCGAAGC).
1. J. J. Skalicky, D. K.
Sukumaran, J. L. Mills, T. Szyperski, J. Am. Chem. Soc., 122 (2000) 3230-3231
2. P. Padrta, R. Štefl, L. Králík, L. Žídek,
V. Sklenář, J. Biomol. NMR, 24 (2002) 1-14