Stability and activity of mesophilic subtilisin E and its thermophilic homolog: Insights from molecular dynamics simulations

Herein we examine the origin of the high-temperature(350 K) behavior of
a thermophilic mutant enzyme (labeled as 5-3H5; see Zhao and Arnold
Prot. Eng. 1999, 12, 47-53) derived from subtilisin E by eight amino
acid substitutions. Through the use of molecular dynamics (MD)
simulations, we have provided molecular-level insights into how point
mutations can affect protein structure and dynamics. From our
simulations we observed a reduced rmsd in several key regions, an
increased overall flexibility, an increase in the number of hydrogen
bonds, and an increase in the number of stabilizing interactions in the
thermophilic system. We also show that it is not a necessary requirement
that thermophilic enzymes be less flexible than their mesophilic
counterparts at low temperatures. However, thermophilic enzymes must
retain their three-dimensional structures and flexibility at high
temperatures in order to retain activity. Furthermore, we have been able
to point out the effects of some of the single substitutions. Even if ii
is not possible yet to give general rules for rational protein design,
we are able to make some predictions on how a protein should be
stabilized in order to be thermophilic. In particular, we suggest that a
promising strategy toward speeding up the design of thermally stable
proteins would be to identify fluxional regions within a. protein
through the use of MD simulations (or suitable experiments). Presumably
these regions allow for autocatalytic reactions to occur and are also
involved in allowing water to gain access to the interior of the protein
and initiate protein unfolding. These fluxional regions could also
adversely affect the positioning of the catalytic machinery, thereby
decreasing catalytic efficiency. Thus, once these locations have been
identified, ``focused'' directed evolution studies could be designed
that stabilize these ``fluxional'' regions.