Separating the thermodynamic impact of non-specific interfacial interactions from confinement effects on protein stability.

The intracellular milieu is incredibly complex, containing myriad degrees of spatial restriction and highly variable interfacial chemistry. It is well established that this environmental complexity impacts diffusive properties, stability, and functional rates of proteins. The relative thermodynamic contributions of confinement versus non-specific interfacial interactions (quinary interactions), however, remain poorly understood. We are using reverse micelles (RMs) as a tool to differentiate between these effects. RMs are spontaneously organizing nanopools of water stabilized by surfactants in bulk non-polar solvent. By using optimized surfactant mixtures, the native states of proteins can be maintained in the RM water core. We have executed folding studies on a wide variety of proteins using varied surfactant mixtures with uniform degrees of confinement. We investigate both equilibrium folding thermodynamics and folding kinetics using various spectroscopic approaches. Our findings begin to unveil the complex relationship between a protein's thermodynamic stability and its local solvating environment, helping to dissect the differential impacts of confinement and quinary interactions.