Protecting Proteins from Desiccation Stress Using Molecular Glasses and Gels.

Faced with desiccation stress, many organisms deploy strategies to maintain the integrity of their cellular components. Amorphous glassy media composed of small molecular solutes or protein gels present general strategies for protecting against drying. We review these strategies and the proposed molecular mechanisms to explain protein protection in a vitreous matrix under conditions of low hydration. We also describe efforts to exploit similar strategies in technological applications for protecting proteins in dry or highly desiccated states. Finally, we outline open questions and possibilities for future explorations.

Not Always Sticky: Specificity of Protein Stabilization by Sugars Is Conferred by Protein-Water Hydrogen Bonds.

Solutes added to buffered solutions directly impact protein folding. Protein stabilization by cosolutes or crowders has been shown to be largely driven by protein-cosolute volume exclusion complemented by chemical and soft interactions. By contrast to previous studies that indicate the invariably destabilizing role of soft protein-sugar attractions, we show here that soft interactions with sugar cosolutes are protein-specific and can be stabilizing or destabilizing. We experimentally follow the folding of two model miniproteins that are only marginally stable but in the presence of sugars and polyols fold into representative and distinct secondary structures: ß-hairpin or α-helix. Our mean-field model reveals that while protein-sugar excluded volume interactions have a similar stabilizing effect on both proteins, the soft interactions add a destabilizing contribution to one miniprotein but further stabilize the other. Using molecular dynamics simulations, we link the soft protein-cosolute interactions to the weakening of direct protein-water hydrogen bonding due to the presence of sugars. Although these weakened hydrogen bonds destabilize both the native and denatured states of the two proteins, the resulting contribution to the folding free energy can be positive or negative depending on the amino acid sequence. This study indicates that the significant variation between proteins in their soft interactions with sugar determines the specific response of different proteins, even to the same sugar.

Resolving the enthalpy of protein stabilization by macromolecular crowding.

Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use 19 F nuclear magnetic resonance spectroscopy to follow the reversible, two-state unfolding thermodynamics of the N-terminal Src homology 3 domain of the Drosophila signal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations. Contrary to most current theories of crowding that emphasize steric protein-crowder interactions as the main driving force for entropically favored stabilization, our experiments show that PEG stabilization is accompanied by significant heat release, and entropy disfavors folding. Using our newly developed model, we find that stabilization by ethylene glycol and small PEGs is driven by favorable binding to the folded state. In contrast, for larger PEGs, chemical or soft PEG-protein interactions do not play a significant role. Instead, folding is favored by excluded volume PEG-protein interactions and an exothermic nonideal mixing contribution from release of confined PEG and water upon folding. Our results indicate that crowding acts through molecular interactions subtler than previously assumed and that interactions between solution components with both the folded and unfolded states must be carefully considered.

ß-Hairpin Miniprotein Stabilization in Trehalose Glass Is Facilitated by an Emergent Compact Non-Native State.

From stem cell freeze-drying to organ storage, considerable recent efforts have been directed toward the development of new preservation technologies. A prominent protein stabilizing strategy involves vitrification in glassy matrices, most notably those formed of sugars such as the biologically relevant preservative trehalose. Here, we compare the folding thermodynamics of a model miniprotein in solution and in the glassy state of the sugars trehalose and glucose. Using synchrotron radiation circular dichroism (SRCD), we find that the same native structure persists in solution and glass. However, upon transition to the glass, a completely different, conformationally restricted unfolded state replaces the disordered denatured state found in solution, potentially inhibiting misfolding. Concomitantly, a large exothermic contribution is observed in glass, exposing the stabilizing effect of interactions with the sugar matrix on the native state. Our results shed light on the mechanism of protein stabilization in sugar glass and should aid in future preservation technologies.

Properties of Aqueous Trehalose Mixtures: Glass Transition and Hydrogen Bonding.

Trehalose is a naturally occurring disaccharide known to remarkably stabilize biomacromolecules in the biologically active state. The stabilizing effect is typically observed over a large concentration range and affects many macromolecules including proteins, lipids, and DNA. Of special interest is the transition from aqueous solution to the dense and highly concentrated glassy state of trehalose that has been implicated in bioadaptation of different organisms toward desiccation stress. Although several mechanisms have been suggested to link the structure of the low water content glass with its action as an exceptional stabilizer, studies are ongoing to resolve which are most pertinent. Specifically, the role that hydrogen bonding plays in the formation of the glass is not well resolved. Here we model aqueous trehalose mixtures over a wide concentration range, using molecular dynamics simulations with two available force fields. Both force fields indicate glass transition temperatures and osmotic pressures that are close to experimental values, particularly at high trehalose contents. We develop and employ a methodology that allows us to analyze the thermodynamics of hydrogen bonds in simulations at different water contents and temperatures. Remarkably, this analysis is able to link the liquid to glass transition with changes in hydrogen bond characteristics. Most notably, the onset of the glassy state can be quantitatively related to the transition from weakly to strongly correlated hydrogen bonds. Our findings should help resolve the properties of the glass and the mechanisms of its formation in the presence of added macromolecules.