Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 70

Table 2.9 Characterization of the active site structure and fluctuations for the C3 and G8
mutants in activated precursor states—cont'd

d-C3U/
G8A

d-C3U/
G8D

d-C3G/
G8C d-WT d-U7C d-C3U d-G8A d-G8I d-G8D

r 3 2

(0.19)

1.88
(0.11)

1.96
(0.17)

2.77
(0.4)

1.96
(0.17)

2.13
(0.24)

- 1.9

(0.13)

1.94
(0.15)

y 3 163.7

(9)

164
(8.5)

161.1
(9.7)

106.8
(11)

160.3
(10.5)

162.8
(9.6)

- 156.1

(10.4)

162.2
(9.9)

This table lists key structural indexes fluctuations for the C3 and G8 mutants, along with the
control mutant U7C in activated precursor states. Data analysis was performed over the last 65 ns
of each simulation with a 10 ps sampling frequency. Distance and angles ( Fig. 2.10 )arein ˚ and
degrees, respectively. SDs are listed in parentheses. Boldface font is used to highlight key quantities
that are significantly altered with respect to the WT simulation upon mutation and that are discussed
in the text. F is the in-line fitness index. 150 The r NN distance between nucleobases in the 3 and 8 posi-
tions. rHB and y HB are the hydrogen bond length and angle for the general base step; defined by G12:
N1 d C17:HO2 0 d C17:O2 0 . r HA and y HA are the hydrogen bond length and angle for the general acid
step; defined by C1.1:O5 0 d G8:HO2 0 d G8:O2 0 . The hydrogen bond contact percentage for the
above entries, defined as the percentage of the snapshots in which r 3.0 ˚ and y 120 . The hydro-
gen bond contact percentage for the above entries, defined as the percentage of the snapshots in which
r 2.5 ˚ and y 150 .

2.1 ˚ ), the distance between
the A9 and scissile phosphates ( d 0 ) in the reactant state is around 4.3 ˚ as
in the crystal structure, and the activated precursor maintains in-line fitness
comparable to the WT simulation and significant hydrogen bonding
between the general acid and the leaving group. In addition, the base stac-
king interactions are very similar between the WT and U7C simulations
( Table 2.12 ).
Perhaps, the most notable difference is that the U7C simulation of the
reactant state does not exhibit a strong hydrogen bond between the
general acid and leaving group ( r HA ¼ 3.41 ˚ in the U7C simulation,
whereas the corresponding value is 2.75 ˚ in the WT simulation). How-
ever, the general acid step occurs at a point farther along the reaction coor-
dinate from the reactant state, and examination of the hydrogen bond of the
general acid in the activated precursor state indicates a comparable, slightly
stronger hydrogen bond interaction ( r HA

the nucleophile in the reactant state ( r HB ¼

2.50 and 2.61 ˚ in the U7C and
WT activated precursor simulations, respectively). Overall, the U7C
simulation results indicate very comparable integrity of the active site,
in-line fitness, and positioning of the general base and acid that are condu-
cive for catalysis.

¼