Separating confinement effects from interfacial interactions for the model protein myoglobin using encapsulation in reverse micelles of varying compositions.

The vast majority of thermodynamic studies of protein stability have been executed in dilute aqueous solution. Conditions inside cells, however, present considerable confinement and a wide variety of interfacial interactions. Here, we use reverse micelles to better understand the interplay between confinement, expected to stabilize proteins, and interfacial interactions, expected to destabilize proteins. Reverse micelles are spontaneously organizing complexes composed of surfactants enclosing a nanoscale water pool dissolved in a nonpolar solvent. Surfactants like decylmonoacyl glycerol (10MAG) and lauryldimethylamino-N-oxide (LDAO) or cetyltrimethylammonium bromide (CTAB) with hexanol have been proven to effectively encapsulate proteins in confined environments while preserving their native conformations. We used the model protein myoglobin to examine the thermodynamic impacts of a protein that unfolds via a molten globule intermediate. By comparing myoglobin denaturation under two different encapsulation conditions to aqueous solution using two denaturants, our findings show that confined conditions tend to turn the two-state unfolding into a concerted process by reducing the thermodynamic barriers to unfolding. The nature of the surfactant interface plays an important role in determining the extent of this effect. This reduction of the thermodynamic barrier is most clearly seen in CTAB encapsulation, where the required energy is reduced drastically, and transitions occur near simultaneously regardless of denaturant used. The insight gained from this study provides the framework for future experiments to further study the stability and transition states of proteins in confined environments.