It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T 4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A-benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T 4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.protein stability | protein folding and cooperativity | protein hydration | high-pressure NMR | reverse micelle encapsulation

The encapsulation of proteins and nucleic acids within the nanoscale water core of reverse micelles has been used for over three decades as a vehicle for a wide range of investigations including enzymology, the physical chemistry of confined spaces, protein and nucleic acid structural biology, and drug development and delivery. Unfortunately, the static and dynamical aspects of the distribution of water in solutions of reverse micelles complicate the measurement and interpretation of fundamental parameters such as pH. This is a severe disadvantage in the context of (bio)chemical reactions and protein structure and function, which are generally highly sensitive to pH. There is a need to more fully characterize and control the effective pH of the reverse micelle water core. The buffering effect of titratable head groups of the reverse micelle surfactants is found to often be the dominant variable defining the pH of the water core. Methods for measuring the pH of the reverse micelle aqueous interior using one-dimensional 1H and two-dimensional heteronuclear NMR spectroscopy are described. Strategies for setting the effective pH of the reverse micelle water core are demonstrated. The exquisite sensitivity of encapsulated proteins to the surfactant, water content, and pH of the reverse micelle are also addressed. These results highlight the importance of assessing the structural fidelity of the encapsulated protein using multidimensional NMR before embarking upon a detailed structural and biophysical characterization.

Ice-binding proteins (IBPs) depress the freezing point of body fluids below the melting point, resulting in a thermal hysteresis (TH) that prevents freezing of the organism. The potential of these proteins in the medical sector, in cryopreservation, in the frozen food industry, and in agriculture is enormous. We are investigating the mechanism by which IBPs interact with ice surfaces and inhibit ice growth and recrystallization. We have developed novel methods for these studies, including fluorescence microscopy techniques combined with temperature-controlled microfluidic devices. These techniques have enabled the replacement of the IBP solution surrounding an IBP-bound ice crystal by buffer, without losing the bound IBP or the TH activity. Our results show the irreversibility of the protein:ice interactions and the indirect dependence of TH activity on the protein concentration in solution. We found that the dynamics of the interactions with ice vary dramatically between different types of IBPs. From our results and other recent developments a new understanding of the mechanisms by which various IBPs act is emerging. This understanding is critical for the successful use of IBPs in cryobiological applications.