The ensemble of forces that stabilize protein structure and facilitate biological function are intimately linked with the ubiquitous aqueous environment of living systems. As a consequence, biomolecular activity is highly sensitive to the interplay of solvent-protein interactions, and deviation from the native conditions, for example by exposure to increased thermal energy or severe dehydration, results in denaturation and subsequent loss of function. Although certain enzymes can be extracted into non-aqueous solvents without significant loss of activity, there are no known examples of solvent-less (molten) liquids of functional metalloproteins. Here we describe the synthesis and properties of room-temperature solvent-free myoglobin liquids with near-native structure and reversible dioxygen binding ability equivalent to the haem protein under physiological conditions. The realization of room-temperature solvent-free myoglobin liquids with retained function presents novel challenges to existing theories on the role of solvent molecules in structural biology, and should offer new opportunities in protein-based nanoscience and bionanotechnology.
Water molecules play a number of critical roles in enzyme catalysis, including mass transfer of substrates and products, nucleophilicity and proton transfer at the active site, and solvent shell-mediated dynamics for accessing catalytically competent conformations. The pervasiveness of water in enzymolysis therefore raises the question concerning whether biocatalysis can be undertaken in the absence of a protein hydration shell. Lipase-mediated catalysis has been undertaken with reagent-based solvents and lyophilized powders, but there are no examples of molecularly dispersed enzymes that catalyse reactions at sub-solvation levels within solvent-free melts. Here we describe the synthesis, properties and enzyme activity of self-contained reactive biofluids based on solvent-free melts of lipase-polymer surfactant nanoconjugates. Desiccated substrates in liquid (p-nitrophenyl butyrate) or solid (p-nitrophenyl palmitate) form can be mixed or solubilized, respectively, into the enzyme biofluids, and hydrolysed in the solvent-free state. Significantly, the efficiency of product formation increases as the temperature is raised to 150°C.
Isolating solvent effects by studying proteins in a liquid phase devoid of solvent has not been previously possible because freeze-dried protein solids do not melt but thermally degrade. Herein we circumvent this problem by modifying the interactions between myoglobin molecules via a polymer-surfactant coronal layer to produce a solvent-free liquid phase that is thermally stable over a wide temperature range. Using high-resolution synchrotron radiation circular dichroism and UV-Vis spectroscopies we determine the temperature-dependent structure and re-folding behaviour of cationized myoglobin under solvent-free conditions, and show that dehydration and subsequent melting of the nanoconstruct has no significant effect on the protein secondary structure at room temperature. Significantly, the solvent-free liquid myoglobin molecules exhibit hyper-thermophilic behaviour and can be reversibly refolded by cooling from 155 C. We attribute the abnormally high thermal stability and persistence of protein folding to entropic contributions associated with macromolecular crowding and confinement, and propose that re-folding in the absence of a solvent shell is facilitated by the configurational flexibility and molecular interactivity of the polymer surfactant coronal layer.
Nonaqueous biocatalysis is rapidly becoming a desirable tool for chemical and fuel synthesis in both the laboratory and industry. Similarly, ionic liquids are increasingly popular anhydrous reaction media for a number of industrial processes. Consequently, the use of enzymes in ionic liquids as efficient, environment-friendly, commercial biocatalysts is highly attractive. However, issues surrounding the poor solubility and low stability of enzymes in truly anhydrous media remain a significant challenge. Here, we demonstrate for the first time that engineering the surface of a protein to yield protein-polymer surfactant nanoconstructs allows for dissolution of dry protein into dry ionic liquids. Using myoglobin as a model protein, we show that this method can deliver protein molecules with near native structure into both hydrophilic and hydrophobic anhydrous ionic liquids. Remarkably, using temperature-dependent synchrotron radiation circular dichroism spectroscopy to measure half-denaturation temperatures, our results show that protein stability increases by 55 °C in the ionic liquid as compared to aqueous solution, pushing the solution thermal denaturation beyond the boiling point of water. Therefore, the work presented herein could provide a platform for the realization of biocatalysis at high temperatures or in anhydrous solvent systems.
The increasing requirement to produce platform chemicals and fuels from renewable sources means advances in biocatalysis are rapidly becoming a necessity. Biomass is widely used in nature as a source of energy and as chemical building blocks. However, recalcitrance towards traditional chemical processes and solvents provides a significant barrier to widespread utility. Here, by optimizing enzyme solubility in ionic liquids, we have discovered solvent-induced substrate promiscuity of glucosidase, demonstrating an unprecedented example of homogeneous enzyme bioprocessing of cellulose. Specifically, chemical modification of glucosidase for solubilization in ionic liquids can increase thermal stability to up to 137 °C, allowing for enzymatic activity 30 times greater than is possible in aqueous media. These results establish that through a synergistic combination of chemical biology (enzyme modification) and reaction engineering (solvent choice), the biocatalytic capability of enzymes can be intensified: a key step towards the full-scale deployment of industrial biocatalysis.
The observation of biological activity in solvent-free protein-polymer surfactant hybrids challenges the view of aqueous and nonaqueous solvents being unique promoters of protein dynamics linked to function. Here, we combine elastic incoherent neutron scattering and specific deuterium labeling to separately study protein and polymer motions in solvent-free hybrids. Myoglobin motions within the hybrid are found to closely resemble those of a hydrated protein, and motions of the polymer surfactant coating are similar to those of the hydration water, leading to the conclusion that the polymer surfactant coating plasticizes protein structures in a way similar to hydration water.
The thermal denaturation of solvent-free liquid lysozyme at temperatures in excess of 200 °C was studied by synchrotron radiation circular dichroism spectroscopy. Temperature-dependent changes in the secondary structure were used to map the equilibrium denaturation pathway and characterize a reactive β-sheet-rich unfolding intermediate that was stable in the solvent-free liquid phase under anhydrous conditions but which underwent irreversible aggregation in the presence of water. The unfolding intermediate had a transition temperature of 78 °C and was extremely stable to temperature, eventually reaching the fully denatured state at 178 °C. We propose that the three-stage denaturation pathway arises from the decreased stability of the native state due to the absence of any appreciable hydrophobic effect, along with an entropically derived stabilization of the reactive intermediate associated with molecular crowding in the solvent-free liquid.
Solvent-free liquid proteins are a new class of thermally stable hybrid bionanomaterials that are produced by extensive lyophilization of aqueous solutions of protein-polymer surfactant nanoconjugates followed by thermal annealing. The hybrid constructs, which consist of a globular protein core surrounded by a monolayer of electrostatically coupled polymer surfactant molecules, exhibit nativelike structure, function, and backbone dynamics over a large temperature range. Despite the key importance of the polymer surfactant shell, very little is known about the atomistic structure of the corona and how it influences the phase behavior and properties of these novel nanoscale objects. Here we present molecular dynamics simulations of protein-polymer surfactant nanoconjugates consisting of globular cores of myoglobin or lysozyme and demonstrate that the derived structural parameters are highly consistent with experimental values. We show that the coronal layer structure is responsive to the dielectric constant of the medium and that the mobility of the polymer surfactant molecules is significantly hindered in the solvent-free state, providing a basis for the origins of retained protein dynamics in these novel biofluids. Taken together, our results suggest that the extension of molecular dynamics simulations to hybrid nanoscale objects could be of generic value in diverse areas of soft matter chemistry, bioinspired engineering, and biomolecular nanotechnology.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.