Abstract
Molecular dynamics simulations were performed to study thermal stabilization of proteins via electrostatic interactions of ion pairs. Dynamic motions of four ion pairs previously proposed to be important in thermal stability of adenylate kinase from the thermophile Bacillus stearothermophilus were monitored during the simulation. One of the four ion pairs identified in the crystal structure, Lys180-Asp114, was not maintained in close contact suggesting that the ion pair does not contribute to thermal stability. Among the other three ion pairs, the ion pair Arg116-Glu198 was proposed to be the most important for stability. To predict behaviors of the ion pairs when engineered into a mesophilic homologue to increase stability, in silico mutants of adenylate kinase from the mesophile Bacillus subtilis were generated, and their molecular dynamics simulations were carried out. The ion pairs in the mutant simulations displayed similar behaviors to those in the simulation of the thermophilic protein. To validate the results of the simulations experimentally, the same mutants were produced in vitro and their thermal stabilities were measured using differential scanning calorimetry. In agreement with the simulations, the Lys180-Asp114 did not result in any increase in stability by itself or additive effect with other ion pairs, whereas a mutant with the Arg116-Glu198 exhibited the highest stability among the mutants having one of the four ion pairs. These results provide specific knowledge about stability in adenylate kinases and more generally suggest that molecular dynamics simulations can provide valuable information for identifying and engineering ion pairs.
Highlights
Because proteins from organisms living at high temperatures generally have increased stability, they have drawn attention for both practical and fundamental reasons
Dynamic motions of four ion pairs previously proposed to be important in thermal stability of adenylate kinase from the thermophile Bacillus stearothermophilus were monitored during the simulation
These results provide specific knowledge about stability in adenylate kinases and more generally suggest that molecular dynamics simulations can provide valuable information for identifying and engineering ion pairs
Summary
INSIGHTS FROM MOLECULAR DYNAMICS SIMULATIONS OF THERMOPHILIC AND MESOPHILIC HOMOLOGUES*. Molecular dynamics simulations were performed to study thermal stabilization of proteins via electrostatic interactions of ion pairs. To predict behaviors of the ion pairs when engineered into a mesophilic homologue to increase stability, in silico mutants of adenylate kinase from the mesophile Bacillus subtilis were generated, and their molecular dynamics simulations were carried out. Adenylate kinases from the thermophile Bacillus stearothermophilus (AKste) and the mesophile Bacillus subtilis (AKsub) are excellent targets to study protein stabilization They come from organisms in the same genus and have a high. The differences between them most probably result from temperature adaptation unlike other examples where pairs of proteins are evolutionarily distant and differences related to temperature may be substantially masked by random or other genetic drift Their crystal structures have been solved and compared with each other to reveal the molecular mechanism for the increased stability of AKste (20, 21). The AKsub mutants were produced in vitro and tested for their thermal stability to compare the results with those from the MD simulations
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