Chemistry Reference
In-Depth Information
can bridge the knowledge gap between protein and nucleic acid enzymes, and also
provide critical information for metal sensor development in sensing applications.
Many techniques can be used to probe nucleic acid-metal ion interactions.
Most of the approaches were initially designed and applied to study functions and
mechanisms of ribozymes, because these catalytic RNAs are believed to play an
important role in the early stage of life on Earth. One of the most intriguing objec-
tives of nucleic acid enzyme research is the elucidation of structures of the active
enzymes bound to metal ion cofactors. This goal was achieved by means of X-
ray crystallography. Crystal structures of metal ion-ribozyme complexes were
obtained.
56,57
These structures show that catalytic RNAs have complex three-dimen-
sional structures and may contain multiple metal ion binding sites required for
proper folding and activity. Even though X-ray crystallography provides direct visu-
alization of nucleic acid enzyme structures, its application is limited to the solid
crystal phase and often affected by the lack of diffraction quality crystals. Several
other techniques, including nuclear magnetic resonance (NMR) and electron para-
magnetic resonance (EPR) can be employed to study enzyme-metal ion interac-
tions in the solution phase in close to physiological conditions. They often take
advantage of paramagnetic metal ions or atoms on the enzyme, and monitor the
changes in their spin states in magnetic fi elds during metal-enzyme interactions. For
example, DeRose and colleagues used paramagnetic Mn
2+
to substitute native Mg
2+
in a hammerhead model ribozyme.
58
The EPR signal from Mn
2+
was sensitive to the
environment surrounding the metal ion, thus providing small but measurable
changes in the EPR signal during interaction with the enzyme. Specifi cally, binding
of Mn
2+
to a biomolecule induced a small ligand fi eld asymmetry and increased its
rotational correlation time, leading to considerable reduction in the amplitude of
the six-line EPR signal from the bound Mn
2+
. This correlation was utilized to dif-
ferentiate free and bound Mn
2+
in the presence of the ribozyme. Binding affi nity
and the number of bound Mn
2+
ions were determined using titration curves based
on EPR signals. They found that the ribozyme contained four high-affi nity binding
sites for Mn
2+
with a
K
d
of
460 m M.
In a more recent report, Britt, Derose and coworkers employed pulsed EPR tech-
niques of electron spin-echo envelope modulation (ESEEM) and electron spin-echo
electron nuclear double resonance (ESE-ENDOR) for structural analysis of the
paramagnetic metal ion Mn(II) bound to nucleotides and nucleic acids.
59
Spin - echo
EPR techniques allow the analysis of paramagnetic metal ion interactions with
magnetic nuclei of ligands and nearby regions of bound macromolecules.
59
A com-
bined analysis of ESEEM and ESE-ENDOR data provided information on the
number, type and spatial distribution of magnetic nuclei in the neighbourhood of
the Mn
2+
ion.
59
Additionally, the exact metal ion ligation site on the nucleotide and
the hydration number of Mn
2+
in the complex were also determined. In another
related report, Schiemann and coworkers employed a strategy that replaced Mn
2+
ions bound to a Diels-Alder ribozyme with higher-affi nity, but EPR-silent, Cd
2+
ions.
In this way, they were able to characterize each of the fi ve Mn
2+
binding sites indi-
vidually using EPR signals.
60
Given these results, it is clear that EPR and related
techniques provide a powerful approach to highly precise characterization of the
structural features of nucleic acid-metal ion complexes.
∼
4 mM and fi ve other binding sites with a
K
d
of
∼
