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Protein Structure and Hydration Probed by SANS and Osmotic Stress

Christopher Stanley ^* , Susan Krueger ^*, V. Adrian Parsegian  and Donald C. Rau 

^* NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland; and Laboratory of Physical and Structural Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

Correspondence: Address reprint requests to Christopher Stanley, Neutron Scattering Science Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831. E-mail: stanleycb{at}ornl.gov.

Interactions governing protein folding, stability, recognition, and activity are mediated by hydration. Here, we use small-angle neutron scattering coupled with osmotic stress to investigate the hydration of two proteins, lysozyme and guanylate kinase (GK), in the presence of solutes. By taking advantage of the neutron contrast variation that occurs upon addition of these solutes, the number of protein-associated (solute-excluded) water molecules can be estimated from changes in both the zero-angle scattering intensity and the radius of gyration. Poly(ethylene glycol) exclusion varies with molecular weight. This sensitivity can be exploited to probe structural features such as the large internal GK cavity. For GK, small-angle neutron scattering is complemented by isothermal titration calorimetry with osmotic stress to also measure hydration changes accompanying ligand binding. These results provide a framework for studying other biomolecular systems and assemblies using neutron scattering together with osmotic stress.