Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 62

4.3.4 Production simulation
After 10 ns of solvent equilibration, the whole systemwas energy-optimized
and unconstrained dynamics simulation began from 0 K under constant
pressure of 1 atm. The temperature was increased to 298 K at the rate of
1 K/ps and then kept fixed at 298 K. The same equilibration process was
applied for each simulation. At the first 10 ns production simulation,
two harmonic restraints of 20 kcal/mol/ ˚ 2 were added to keep the
Mg 2 þ ion binding to G10.1:N7 and A9:O2P position. Another three har-
monic restraint of 20 kcal/mol/ ˚ 2 were used: the distances between
G8:HO2P and C1.1:O5 0 , and between G12:H1 and C17:O2 0 , were
kept around 1.8 ˚ to ensure the initial hydrogen bonding; the distance
between A9: O2P and C1.1:O2P was kept at 4.3 ˚ (crystal distance). After
10 ns, all restraints were removed. The motions and relaxation of solvent
and counter-ions are notoriously slow to converge in nucleic acid
simulations, 115 and careful equilibration is critical for reliable simulations.
In summary, each simulation was carried out to 300 ns beginning with a total
of 20 ns of equilibration (10 ns of solvent/ion relaxation and 10 ns of solvent
and structure relaxation). Analysis was performed over the last 250 ns with
data collected every 10 ps.
5. COMPUTATIONAL MUTAGENESIS OF KEY RESIDUES
OF HHR
In this section, we report MD simulations to elucidate the origin of
mutational effects in the HHR. In a series of 24 100-ns molecular MD sim-
ulations, we explore the structure and dynamics of eHHR mutants involv-
ing C3, G5, U7, and G8 positions in both the reactant and activated
precursor (deprotonated 2 0 OH) states. Simulations for each mutation are
compared with the wild-type (WT) simulation results. The activated pre-
cursor state, distinguished by the deprotonation of the 2 0 OH group of
C17, is already described in previous sections.
The U7 mutation has been known as a benign mutation, and hence is
used as a control simulation in this study. Single mutations at both the
C3 and G8 positions (C3U, G8A, G8I, and G8D) are explored, where
“D” indicates 2,6-diaminopurine, and “I” indicates inosine. In addition,
double mutants that exhibit a partial rescue effect have been examined,
including the isosteric C3U/G8D and hydrogen bond-preserving C3G/
G8C double mutation. A simulation of C3U/G8D mutations, for which
there currently exists, to our knowledge, no experimental measurement,