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 t hermophilic mutant enzyme (labeled as 5-3H5; see Zhao and Arnold Prot. Eng. 1999, 12, 47-53) derived from subtilisin E by eight amino acid substitutio ns. Through the use of molecular dynamics (MD) simulations, we have provide d molecular-level insights into how point mutations can affect protein stru cture and dynamics. From our simulations we observed a reduced rmsd in seve ral key regions, an increased overall flexibility, an increase in the numbe r of hydrogen bonds, and an increase in the number of stabilizing interacti ons in the thermophilic system. We also show that it is not a necessary req uirement that thermophilic enzymes be less flexible than their mesophilic c ounterparts 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 e ffects 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 thermophi lic. In particular, we suggest that a promising strategy toward speeding up the design of thermally stable proteins would be to identify fluxional reg ions within a. protein through the use of MD simulations (or suitable exper iments). Presumably these regions allow for autocatalytic reactions to occu r 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 d ecreasing catalytic efficiency. Thus, once these locations have been identi fied, "focused" directed evolution studies could be designed that stabilize these "fluxional" regions.