Stability and activity of mesophilic subtilisin E and its thermophilic homolog: Insights from molecular dynamics simulations
This report examines the origin of the high-temperature (250 K) behavior of a thermophilic mutant enzyme (labeled at 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, the authors have provided molecular-level insights into how point mutations can affect protein structure and dynamics. From simulations the authors 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. It was shown 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, the authors have been able to point out the effects of some of the single substitutions. Even if it is not possible yet to give general rules for rational protein design, the authors are able to make some predictions on how a protein should be stabilized in order to be thermophilic. In particular, the authors 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.
- Research Organization:
- Pennsylvania State Univ., University Park, PA (US); Ist. di Biocatalisi e Riconoscimento Molecolare CNR, Milano (IT)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG02-96ER62270
- OSTI ID:
- 20000042
- Journal Information:
- Journal of the American Chemical Society, Vol. 121, Issue 29; Other Information: PBD: 28 Jul 1999; ISSN 0002-7863
- Country of Publication:
- United States
- Language:
- English
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