Proteins: Ssp DnaE Intein - Multiscale Modeling in Biomechanics and Mechanobiology - page 10

Fig. 1.3 Gel of WT, T69A,
and T69S mutations. WT
corresponds to the native wild
type intein without mutations,
while T69A and T69S
correspond to mutations of
threonine to alanine and
serine, respectively
we believe this provides an estimate in the reduction of the reaction barrier due to
threonine. To examine the cause behind the difference in energy barriers, we examine
the charges on the sulfur of the catalytic cysteine and the carbonyl oxygen on the
glycine. We used the Natural Population Analysis (NPA) [ 40 ] scheme to obtain
the atomic charges for the system. We see that with the presence of the threonine
nearby, the sulfur charge on the catalytic cysteine is reduced from that of the system
containing alanine. Furthermore, we believe that the solvent is causing a charge
screening effect between the sulfur and oxygen. Thus, the screening effect combined
with the reduced atomic charges on the sulfur allow for an easier transition of the
sulfur to bond with the carbon during the N-S acyl shift, which is represented by a
lower reaction barrier. This conclusion is supported by experimental results shown in
Fig. 1.3 , which show that upon mutation of threonine to alanine, the splicing reaction
is halted. Furthermore, when threonine is mutated to serine, the reaction does not
proceed as quickly as with the wild type threonine, however it is not completely
halted like in the case of alanine. This lends further support to the conclusion that
the hydroxyl side chain bonding with the glycine at the -1 position is affecting the
intein splicing process.
In conclusion, we have used multiscale modeling to investigate the DnaE intein
that was trapped in its precursor state that had previously never before been observed.
Using a mixed QM/MM method to incorporate the accuracy of fully quantum
mechanical density functional theory combined with inexpensive molecular mechan-
ics methods for large structures, we were able to reliably investigate the source of a
distortion found at Gly-1. We determined that the source is due to a hydrogen bond-
ing interaction between Gly-1 and the nearby Thr69. Furthermore, we were able to
computationally predict the structure of the system when threonine was mutated to
alanine, which was then later verified by experiment. By investigating the effects
of threonine and alanine on the catalytic region between Cys1 and Gly-1, we found
that threonine serves to enhance the reaction rate by lowering the reaction barrier
of the N-S acyl shift. We have also shown that when alanine is present in place of
threonine, the reaction barrier is far greater and splicing ceases. This study not only
provides a greater insight into the fundamental understanding of protein splicing, but
also could possibly have implications in reactions that require isolation of precursor
structures.Although still a relatively new method of investigation, multiscale mod-
eling may help open the door to new and larger studies theoretically that will help
further our knowledge of experiments.