Static and Dynamic Water Molecules in Cu,Zn Superoxide Dismutase M. Falconi, 1 M. Brunelli, 2 A. Pesce, 3 M. Ferrario, 2 M. Bolognesi, 3 and A. Desideri 1 * 1 INFM (National Institute for the Physics of the Matter) and Department of Biology University of Rome “Tor Vergata”, Rome, Italy 2 INFM (National Institute for the Physics of the Matter)-S3 and Department of Physics, University of Modena and Reggio Emilia, Modena, Italy 3 INFM (National Institute for the Physics of the Matter) Department of Physics and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy ABSTRACT Understanding protein hydration is a crucial, and often underestimated issue, in unraveling protein function. Molecular dynamics (MD) computer simulation can provide a micro- scopic description of the water behavior. We have applied such a simulative approach to dimeric Pho- tobacterium leiognathi Cu,Zn superoxide dismutase, comparing the water molecule sites determined using 1.0 ns MD simulation with those detected by X-ray crystallography. Of the water molecules de- tected by the two techniques, 20% fall at common sites. These are evenly distributed over the protein surface and located around crevices, which repre- sent the preferred hydration sites. The water mean residence time, estimated by means of a survival probability function on a given protein hydration shell, is relatively short and increases for low acces- sibility sites constituted by polar atoms. Water mol- ecules trapped in the dimeric protein intersubunit cavity, as identified in the crystal structure, display a trajectory mainly confined within the cavity. The simulation shows that these water molecules are characterized by relatively short residence times, because they continuously change from one site to another within the cavity, thus hinting at the ab- sence of any relationship between spatial and tempo- ral order for solvent molecules in proximity of protein surface. Proteins 2003;51:607– 615. © 2003 Wiley-Liss, Inc. Key words: X-ray; molecular dynamics; water den- sity peaks; water residence times; hydra- tion sites; protein–water interactions INTRODUCTION Water in close proximity to the protein surface is fundamental to protein folding, stability, recognition, and activity. The full understanding of solvent–protein interac- tions is a key issue in the comprehension of the protein functionality. Interactions between amino acid residues and their aqueous–protein environments together first determine protein folding, then mediate intermolecular interactions. On the other hand, not only water influences protein mobility, folding, and function, but also proteins can modify water structure and dynamics. 1 Water mol- ecules in protein solutions may be broadly classified into three categories 2 : 1. Strongly bound internal water 2. Water molecules that interact with the protein surface 3. Bulk water. Bound water molecules occupying internal cavities and deep clefts can be identified crystallographically. 3 Such water molecules, which are extensively involved in the protein–solvent H-bonding, often play a structural role. On the other hand, surface water, usually called hydration water, may exhibit a heterogeneous behavior because of its interaction with the solvent-exposed protein atoms having different chemical character and roughness. Finally, wa- ter that is not in direct contact with the protein, continu- ously exchanging with surface water, reveals properties that approach those of bulk water to the degree that solvent molecules at increasing distance from the protein surface are taken into account. 4 Information on the water position around a protein is provided by X-ray or neutron diffraction experiments on protein crystals, 5–7 with both techniques revealing the favored average positions occupied by water molecules. Dynamic information on water behavior is provided by NMR spectroscopy, which can distinguish between the dynamics of surface and internal waters in a protein, 8,9 and by inelastic neutron scattering studies of H 2 O- hydrated powders of fully deuterated proteins, which have provided evidence that water undergoes jump diffusion on the protein surface. 10 –12 Molecular dynamics (MD) simula- tion is also a powerful tool to describe protein–solvent interaction, because it provides a microscopic description of the protein–water system on the nanosecond time scale, allowing either the evaluation of the preferential water hydration sites 13,14 or the evaluation of the mean resi- Grant sponsor: MURST COFIN 2000 project. *Correspondence to: A. Desideri, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, 00133 Rome, Italy. E-mail: desideri@uniroma2.it Received 19 September 2002; Accepted 12 December 2002 PROTEINS: Structure, Function, and Genetics 51:607– 615 (2003) © 2003 WILEY-LISS, INC.