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Applied Magnetic Resonance

, Volume 31, Issue 3–4, pp 543–552 | Cite as

W-band31P-ENDOR on the high-affinity Mn2+ binding site in the minimal and tertiary stabilized hammerhead ribozymes

  • O. Schiemann
  • R. Carmieli
  • D. Goldfarb
Article

Abstract

The catalytic activity of the tertiary stabilized hammerhead ribozyme (tsHHRz) is by three orders of magnitude higher than the one of the long-known minimal construct (mHHRz). This gives rise to the question whether the single high-affinity manganese(II) binding site present in both ribozymes is located closer to the cleavage site and the transition state in the tsHHRz than in the mHHRz, which would make a direct involvement of this metal(II) ion in the bond-breaking step more likely. Here, we used W-band31P-Davies-ENDOR (electron-nuclear double resonance) to complement earlier reported14N-ESEEM/HYSCORE (electron spin echo envelope modulation/hyperfine sublevel correlation) studies. The31P-ENDOR spectrum of the mHHRz revealed a doublet with a splitting of 8.4(±0.5) MHz but unresolved hyperfine anisotropy. Such a large splitting indicates an inner-sphere coordination of a phosphate backbone group with a significant amount of spin density on the phosphorous nucleus. This is in good agreement with the31P isotropic hyperfine constant,A iso(31P), of +7.8 MHz obtained by density functional theory calculations on the structure of the Mn2+ binding site as found in crystals of the same ribozyme. This supports the idea that the structure and location of the binding site in the mHHRz is in frozen buffer similar to that found in the crystal. Since the W-band ENDOR spectrum of the tsHHRz also shows a31P splitting of 8.4(±0.5) MHz, the local structures of both binding sites appear to be similar, which agrees with the coincidence of the14N data. An involvement of the high-affinity Mn2+ ion in the catalytic step seems therefore unlikely.

Keywords

Electron Paramagnetic Resonance Electron Paramagnetic Resonance Spectrum Density Functional Theory Calculation ENDOR Spectrum Hammerhead Ribozyme 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer 2007

Authors and Affiliations

  1. 1.Institute of Physical and Theoretical ChemistryJ. W. Goethe UniversityFrankfurtGermany
  2. 2.Department of Chemical PhysicsWeizmann Institute of ScienceRehovotIsrael

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