Modulation of Environmental Dynamics at the Active Site and Activity of an Enzyme under Nanoscopic Confinement: Subtilisin Carlsberg in Anionic AOT Reverse Micelle

Hydration dynamics plays a crucial role in determining the structure, function, dynamics, and stability of an enzyme. These dynamics involve the trapped-water motions within small distance along with the total protein dynamics. However, the exact molecular basis for the induction of enzyme function by water dynamics is still remain unclear. Here, we have studied both enzymatic activity and environmental dynamics at the active site of an enzyme, Subtilisin Carlsberg (SC), under confined environment of the reverse micelle (RM) retaining the structural integrity of the protein. Kinetic measurements show that enzymatic activity increases with increasing the water content of the RM. The picosecond-resolved fluorescence Stokes shift studies indicate faster hydration dynamics at the active site of the enzyme with increasing the water content in the RM (<i>w</i>₀ values). Temperature-dependent hydration dynamics studies demonstrate the increased flexibility of the protein at higher temperature under confinement. From temperature-dependent solvation dynamics study, we have also calculated the activation energy that has to be overcome for full orientational freedom to the water molecules from bound to free-state. The results presented here establish a correlation between the enzymatic activity and dynamics of hydration of the encapsulated protein SC in cell-like confined environment within the structural integrity of the enzyme

Microstructure, Morphology, and Ultrafast Dynamics of a Novel Edible Microemulsion

An edible microemulsion (ME) composed of Tween 80/butyl lactate/isopropyl myristate (IPM)/water has been formulated. Pseudoternary phase diagram of the system contains a large single isotropic region. The phase behavior of the system is also studied at low pH (2.6) and in 0.9% NaCl solution. Conductivity, viscosity, ultrasonic velocity, and compressibility studies find consistent results in the structural transition (from water-in-oil (w/o) to bicontinuous, and from bicontinuous to oil-in-water (o/w)) behavior of the ME. Dynamic light scattering studies reveal the size of the MEs. The absorption and steady state emission spectra of 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM) successfully probe the polarity of the ME at its solvation shell and shows the efficacy of hosting model drug molecules. The rotational anisotropy of the dye has been studied to ascertain the geometrical restriction of the probe molecule. Picosecond-resolved fluorescence spectroscopy applies well to study the relaxation dynamics of water in the solvation shell of the MEs. The study finds strong correlation in the relaxation dynamics of water with the structure of host assembly and offers an edible ME system which could act as a potential drug delivery system and nontoxic nanotemplate for other applications