Proteins function in crowded aqueous environments interacting with a diverse range of compounds, among them dissolved ions. These interactions are water mediated. In the present study we combine field dependent NMR relaxation (NMRD) and theory to probe water dynamics at the surface of protein in concentrated aqueous solutions of hen egg-white lysozyme (LZM) and bovine serum albumin (BSA). The experiments reveal that presence of salts (NaCl or NaI) leads to an opposite ion-specific response for the two proteins: an addition of salt to LZM solutions increases water relaxation rates with respect to the salt-free case, while for BSA solutions a decrease is observed. The magnitude of the change depends on the ion identity. The developed model accounts for the non-Lorentzian shape of the NMRD profiles and reproduces the experimental data over four decades in Larmor frequency (10 kHz to 110 MHz). It is applicable 1

Ion-specific effects at the protein surface are investigated here in light of the changes they infer to surface water dynamics, as observed by 1H NMR relaxation (at 20 MHz). Two well-known proteins, hen egg-white lysozyme (LZM) and bovine serum albumin (BSA), show qualitatively opposite trends in the transverse relaxation rate, R2(1H), along a series of different monovalent salt anions in the solution. Presence of salt ions increases R2(1H) in the case of lysozyme and diminishes it in the case of BSA. The effect magnifies for larger and more polarizable ions. The same contrasting effect between the two proteins is observed for protein-solvent proton exchange. This hints at subtle effects ion-binding might have on the accessibility of water surface sites on the protein. We suggest that the combination of the density of surface charge residues and surface roughness, at the atomic scale, dictates the response to the presence of salt ions and is proper to each protein. Further, a dramatic increase in R2(1H) is found to correlate closely with the formation of protein aggregates. The same ordering of salts in their ability to aggregate lysozyme, as seen previously by cloud point measurements, is reproduced here by R2(1H). 1H NMR relaxation data is supplemented by 35Cl and 14N NMR relaxation for selected salt ions to probe the ion-binding itself.

The effects of additions of low-molecular-mass salts on the properties of aqueous lysozyme solutions are examined by using the cloud-point temperature, T cloud , measurements. Mixtures of protein, buffer, and simple salt in water are studied at pH = 6.8 (phosphate buffer) and pH = 4.6 (acetate buffer). We show that an addition of buffer in the amount above I buffer = 0.6 mol dm −3 does not affect the T cloud values. However, by replacing a certain amount of the buffer electrolyte by another salt, keeping the total ionic strength constant, we can significantly change the cloud-point temperature. All the salts de-stabilize the solution and the magnitude of the effect depends on the nature of the salt. Experimental results are analyzed within the framework of the onecomponent model, which treats the protein-protein interaction as highly directional and of short-range. We use this approach to predict the second virial coefficients, and liquid-liquid phase diagrams under conditions, where T cloud is determined experimentally.

Isothermal titration calorimetry was used to determine the temperature and salt concentration dependence of the enthalpy of mixing, ΔmixH, of bovine serum albumin (BSA) in aqueous buffer solutions with several low molecular weight salts. Three buffers were used: acetate (pH = 4.0), MOPS (7.2), and borate (9.2). Since the isoionic point of BSA is at pI ≈ 4.7, the net charge of BSA in acetate buffer was positive (≈ +20), while in the other two buffer solutions it was negative (≈ −15 in MOPS and ≈ −25 in borate). The majority of the recorded heat effects were exothermic, while only at pH = 9.2 a weak endothermic effect upon mixing BSA with LiCl, NaCl, and KCl was observed. For all buffer solutions the absolute values of ΔmixH of sodium salts followed the order: NaCl < NaBr < NaNO3 < NaI < NaSCN, which is the reverse Hofmeister series for anions. The magnitude of the effects was the largest in acetate buffer and decreased with an increasing pH value of the solution. While the effect of varying the anion of the added salts was strongly pronounced at all pH values, the effect of the cation (LiCl, NaCl, KCl, RbCl and CsCl salts) was weak. The most interesting feature of the results obtained for pH > pI was the fact that ΔmixH were considerably more sensitive to the anion (co-ion to the net BSA charge) than to the cation species. This indicated that anions interacted quite strongly with the BSA even at pH values where the net charge of the protein was negative. We showed that ΔmixH at high addition of salts correlated well with the enthalpy of hydration of the corresponding salt anion. This finding suggested, consistently with some previous studies, that a part of the exothermic contribution to ΔmixH originated from the hydration changes upon the protein–salt interaction. Theoretical analysis, based on the primitive model of highly asymmetric electrolyte solutions solved within the mean spherical approximation, was used to estimate Coulomb effects upon mixing.