A molecular dynamics study of the effect of carbon tetrachloride on enzyme structure and dynamics: subtilisin

Developing enzymes that are functional in highly concentrated halocarbon solutions, such as carbon tetrachloride, may prove useful in the development of new strategies for environmental remediation and monitoring of pollutant plumes, as well as in developing 'green' processes. Doing so will require gaining an understanding of the underlying structural and dynamic effects on enzymes induced by such solvents. Herein we report a 714 ps molecular dynamics simulation of the enzyme subtilisin Carlsberg and its waters of crystallization in a periodic box of carbon tetrachloride. The crystal structure from aqueous solution was used as the starting structure for our simulation using the AMBER program and forcefield. The calculated time-averaged structure is similar to the aqueous X-ray structure except for significant differences in loop (or turn) regions, resulting in many extra intra-protein hydrogen bonding interactions. Since carbon tetrachloride is a non-polar solvent and cannot interact strongly with the protein and water molecules, the water molecules stay very close to the protein surface throughout the simulation. The mobility of most of the waters was therefore very low. A few water molecules underwent significant lateral motion during the simulation, but never wandered far from the protein surface. Waters were either hydrogen bonded to protein polar groups, other water and/or counterions. Some of the surface waters participated in the formation of water-mediated hydrogen bonding networks. The increase in total number of intra-protein hydrogen bonds and the formation of water-mediated hydrogen bonding networks in carbon tetrachloride is consistent with the generally observed increase in thermostability and reduced flexibility of proteins in non-aqueous solutions. Several possible carbon tetrachloride binding sites on the protein surface are predicted.