Many organisms accumulate low molecular weight substances known as osmolytes when they experience environmental water stress. The main classes of osmolytes are sugars, polyhydric alcohols, amino acids and their derivatives, and methylamines, and all are known to be protein stabilizers. However, marine cartilaginous fishes and the coelacanth use, as osmolytes, a combination of urea and methylamines, i.e., a denaturant and a stabilizer, in a 2:1 molar ratio. Preferential binding and thermal denaturation measurements in the presence of each cosolvent separately and in their mixtures have been carried out using ribonuclease T1 (RNase T1) as the protein. At a 2:1 molar ratio of urea and trimethylamine N-oxide (TMAO), the effects of the two cosolvents on the transition temperature (Tm) were found to be essentially the algebraic sum of their effects when used individually. Preferential interaction measurements of urea, TMAO and urea in its 2:1 molar ratio mixture with TMAO, have shown that the presence of TMAO has no effect on the interaction of urea with the protein in either the native or the unfolded (reduced carboxymethylated RNase T1) state. The preferential interaction of TMAO in the presence of urea could not be measured for technical reasons. Calculations of transfer free energy in the two end states of the denaturation reaction have shown that 2 M urea destabilizes RNase T1 by 3.8 +/- 0.3 kcal/mol whether 1 M TMAO is present or not. The contribution of 1 M TMAO to stabilization is calculated to be 3.1 kcal/mol in the presence of 2 M urea and is measured to be 2.7 kcal/mol in its absence.

Surface tension measurements were carried out at 20 degrees C by a capillary drop-weight method on aqueous solutions of sodium glutamate (NaGlu), lysine hydrochloride (LysHCl), potassium aspartate (KAsp), arginine hydrochloride (ArgHCl), lysylglutamate (LysGlu), argininylglutamate (ArgGlu), guanidinium sulfate, trehalose, trimethylamine N-oxide (TMAO), dimethyl sulfoxide, 2-methyl-2,4-pentanediol (hexylene glycol), and poly(ethylene glycol)s of molecular weights 200, 400, 600, and 1000. All of the salts and the sugar increased the surface tension of water, while the last four compounds decreased it, with 2-methyl-2,4-pentanediol lowering it most effectively and TMAO being the least effective. The preferential hydration of bovine serum albumin (BSA) and lysozyme was measured in KAsp, ArgHCl, LysGlu, and ArgGlu. The high values of preferential hydration found in all cases, except for BSA in ArgHCl, suggest that they should stabilize protein structure, as had been found for lysine hydrochloride and monosodium glutamate [Arakawa, T., & Timasheff, S. N. (1984) J. Biol. Chem. 259, 4979-4986]. A correlation was found for both BSA and lysozyme in KAsp, NaGlu, LysHCl, ArgGlu, and LysGlu between the surface tension effect and the observed preferential interactions, indicating that the change in the surface free energy of the protein-containing cavity due to the surface tension increase for water by these amino acid salts contributes dominantly to the observed increase in the chemical potential of the protein by their addition. The lack of a correlation observed for BSA, but not lysozyme, in ArgHCl at low concentrations where preferential binding is close to zero suggests, however, that the surface tension effect is not the sole factor involved in the protein-solvent interactions in these amino acid salts. Binding of ArgHCl to BSA, probably through hydrogen bonds between the Arg guanidinium group and peptide bonds, was proposed to occur, the affinity of Arg+ being reduced by electrostatic repulsion when proteins carry a net positive charge, such as is the case with lysozyme. Since the four organic solvent additives also lead to protein preferential hydration, no correlation exists between their preferential interactions and the surface free energy perturbation. Therefore, in their case, the preferential hydration must be ascribed to other factors that overcome the preferential binding expected from the Gibbs adsorption isotherm. The surface tension results, however, are consistent with the binding of the organic solvents to proteins through hydrophobic interactions, explaining, at least in part, the observed concentration dependence of the interactions.

The stabilization of proteins by a variety of co-solvents can be related to their property of increasing the surface tension of water. It is demonstrated that, during the thermal unfolding of proteins, this increase of the surface tension can be overcome by the increase in the temperature of the solution at the midpoint of the transition, T,, and the weak binding of co-solvent molecules. Three such co-solvents were studied: trehalose, lysine hydrochloride (LysHCl), and arginine hydrochloride (ArgHC1). Trehalose and LysHCl increase the midpoint of T,. The increase of the surface tension by addition of trehalose is completely compensated by its decrease due to the increase in T,. However, for LysHC1, the increase of the surface tension by the co-solvent is partly reduced by its binding to the protein. Later studies on the preferential interaction2 of proteins with a variety of co-solvents have identified the surface tension ef-'The term preferential interaction refers to the net interaction between the protein and the solvent components, water and the co-solvent, as measured by an equilibrium thermodynamic approach, such as dialysis equilibrium. When the interactions are weak, as is the case with co-solvents, such as sugars, glycerol, amino acids, or urea, the measured quantity is the net balance between the weak interactions (binding) of water and co-solvent molecules to the protein over its entire surface. If, on average, the affinity of protein surface loci for the co-solvent is greater than that for water, the dialysis equilibrium experiment will result in an excess of co-solvent at the protein surface over its concentration in the bulk solvent, and the measured binding stoichiometry will be positive, which means that co-solvent is preferentially bound relative to water. In that case, the "preferential binding" will be positive. In the converse case, in which, on average, the affinity of the protein surface loci is greater for water than for co-solvent molecules, there will be an excess of water at the protein surface over its concentration in the 372

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Energy transferred via thermal radiation between two surfaces separated by nanometer distances can be much larger than the blackbody limit. However, realizing a scalable platform that utilizes this near-field energy exchange mechanism to generate electricity remains a challenge. Here, we present a fully integrated, reconfigurable and scalable platform operating in the near-field regime that performs controlled heat extraction and energy recycling. Our platform relies on an integrated nano-electromechanical system that enables precise positioning of a thermal emitter within nanometer distances from a room-temperature germanium photodetector to form a thermo-photovoltaic cell. We demonstrate over an order of magnitude enhancement of power generation (P gen~1 .25 μWcm −2) in our thermophotovoltaic cell by actively tuning the gap between a hot-emitter (T E~8 80 K) and the cold photodetector (T D~3 00 K) from~500 nm down to~100 nm. Our nanoelectromechanical system consumes negligible tuning power (P gen /P NEMS~1 0 4) and relies on scalable silicon-based process technologies.