59th Annual Report on Research 2014

1) DMSO enhancement: DMSO (dimethyl sulfoxide) is a small dipolar aprotic molecule, and can be miscible with both polar and nonpolar solvents. It has an intriguing effect on enzyme activity. Experiments reported the activities of a wide variety of lipases can be enhanced and even optimized with a moderate concentration of DMSO. We have set up and performed short simulations in the first year, now we extend to longer (100 ns) simulations of the bacterium lipase from P. aeruginosa in different concentrations of DMSO . Six different concentrations of DMSO were created (roughly 0%, 15%, 30%, 45%, 60%, 100% w/w). Since DMSO and water are miscible, we have developed a fast premixing procedure to save the solvent equilibration time. The premixing procedure can be generally applicable to the simulation setups of other organic solvent mixtures. The gating motion of the lipase as a function of time is shown in Figure 1. An open gate (measured by a gorge radius larger than ~3 Angstroms) indicates the enzyme is active and can accept the substrate. We can see the gating motion is affected by DMSO concentration. Increasing the concentration of DMSO(15-45% w/w) enhances gating motion, and the gating motions are decreased at higher concentration. Thus, the activity enhancement reported in experiments may come from the protein conformational dynamics as a direct result of DMSO-protein interactions.

2) Pressure activation: Another interesting aspect is the reported activation of the enzyme at an elevated pressure. Pressure perturbation has been reported to have different levels of effects on hydrophobic vs hydrophilic interactions. Although it does not perturb the same way as a water-oil interface, it nevertheless assists us with understanding how interfacial proteins work. There is experimental evidence of lipase being more active at high pressure. However, the non-monotonous behavior and somewhat complicated and indirect experimental setups still hinder a quantitative understanding of the effect of pressure on gating. Again, we employed the aforementioned bacterium lipase, solvated it in aqueous solution, and subjected it to different hydrostatic pressures (1, 1k, 2.5k, 5k, 7.5k,10k, and 15k bar). As seen in Figure 2, we demonstrate directly that lipase gating can be activated at elevated pressures. Here, we observed an open-gate conformation at intermediately high pressure of 5 kbar. At a very high pressure of 15k bar, the gate is closed again.

3) Analysis of the gating motion at the level of residue-residue contact: We also developed a new two-pronged method to study the gating motions of lipase. The first step involves a rigorous data reduction scheme that identifies the dynamic contacts of the system. Next, we perform principal component analysis (PCA) on these residue-residue contacts (rather than using the traditional Cartesian coordinate PCA approach) to further identify the most important collective mode (dynamics of contact changes), so called principal components;. This method has been applied to both pressure effects and DMSO effects on lipases. As seen in Figure 3 (a) and (b), PC1 and PC2 are shown in the contact map format, while the conformations are projected to these two most dominating PCs for the lipase gating activated by high pressures. Here, PC2 captures the effect of gate opening due to the ordering of the alpha 5 region of lipase, while PC1 shows the overall increase of the compactness due to high pressure.