Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 82

not observe for the eHHR an equilibrium between proposed active and
inactive states that involve changes in stem contacts, as has been inferred
from biochemical studies. 149
Recent time-resolved NMR spectroscopic investigations have indicated
that an ensemble of conformations sensitive to the presence of Mg 2 þ are
accessible to mHHR, and that dynamic transitions localized around the cat-
alytic core occur on the ms timescale. 152 This timescale extends beyond that
which has been probed in the current work for the eHHR. Nonetheless, it
remains important to address the question of dynamic conformational equi-
librium and its role in catalysis in future work.
The present molecular simulation results provide insight into the origin
of mutational effects in the eHHR that are not easily derived from available
experimental structural data. We report results of MD simulations of the
native and mutated eHHR in the reactant state and in an activated precursor
state. A key result from this work is that in performing computational muta-
genesis simulations of ribozymes, one must consider timescales greater than
30 ns for relaxation of mutant structures and in some instances look beyond
the reactant state along the catalytic reaction coordinate in order to reconcile
the origin of mutational effects. The present study makes predictions and
offers new insights into the understanding of the HHR mechanism as inter-
preted through mutational data.
5.3. Simulation setup and protocol
MD simulations were set up with protocols identical to that described in
Section 4.3 . The mutants are generated from the VMD package. 154
6. CONCLUSION
In this chapter, we summarized our progress toward understanding
HHR catalysis through a multiscale simulation strategy that employs MD
simulations with classical MM and combined QM/MM potentials, using
linear-scaling electrostatic methods and specialized MM residues and semi-
empirical QMmodels derived from density-functional calculations of phos-
phoryl transfer reactions.
So far, simulations collectively paint a picture of HHR catalysis that
includes a novel role for a catalytic metal ion: the HHR first folds to form
an electrostatic negative pocket to recruit a threshold occupation of cationic
charge, either an Mg 2 þ ion or multiple monovalent ions when Mg 2 þ ions
are not present. The position and coordination pattern of these ions are