Protein hydration in solution: experimental observation by x-ray and neutron scattering

Abstract

The structure of the protein-solvent interface is the subject of controversy in theoretical studies and requires direct experimental characterization. Three proteins with known atomic resolution crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H2O and D2O solutions. The analysis of the protein-solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an average density approximately 10% larger than that of the bulk solvent in the conditions studied. Comparisons with the results of other studies suggest that this may be a general property of aqueous interfaces.

Figure 1

Relationship among the scattering densities of the bulk solvent, protein, and protein–solvent interface for x-rays and neutrons in H₂O and D₂O. (1) Bulk solvent, (2) a shell with density 20% above that of the bulk solvent, (3) mobility of the side chains on the protein surface; scattering density in the interface is drawn in the middle between those of protein and of bulk; (4) protein. Scattering density of protein in D₂O is larger than that in H₂O because of H/D exchange.

Figure 2

Fit to the experimental data by crysol and cryson for lysozyme (a), TR (b), and R1 (c). Symbols represent the experimental curves; solid lines represent the best fits calculated from the atomic models with the hydration shell. Fits (1)–(3) correspond to x-rays, neutrons in H₂O, and neutrons in D₂O, respectively. Arrows indicate the onset of the first shoulder for each curve.

Figure 3

Initial portions of the experimental curves from lysozyme (notations are as in Fig. 2) normalized to the same value of the forward scattering I(0).

Figure 4

Radius of gyration of lysozyme as function of concentration for different solvents: SANS in H₂O, 150 mM NaCl (1); SANS in D₂O, no salt (2); SAXS in H₂O and in D₂O, 150 mM NaCl (3); SAXS in H₂O and in D₂O, no salt (4).