Water molecules are bound to proteins internally and externally. Some water molecules occasionally occupy small internal cavities in the protein structure, and are hydrogen-bonded to peptide bonds and side chains of the protein and often to a prosthetic group, or cofactor, within the protein. The protein surface islarge and consists of a mosaic of polar and nonpolar amino acids, and it binds a large number of water molecules, i.e., it is hydrated, from the surrounding environment. As described in the previous section, water molecules trapped in the interior of protein molecules are bound more tightly to hydrogen-bonding donors and acceptors because of a lower dielectric constant.
Solvent around the protein surface clearly has a general role in hydrating peptide and side chains but might be expected to be rather mobile and non-specific in its interactions. Well-ordered water mole-cules can make significant contributions to protein stability. One water molecule can hydrogen-bond to two groups distant in the primary structure on a protein molecule, acting as a bridge between these groups. Such a water molecule may be highly restricted in motion, and can contribute to the stability, at least locally, of the protein, since such tight binding may exist only when these groups assume the proper configuration to accommodate a water molecule that is present only in the native state of the protein. Such hydration can also decrease the flexibility of the groups involved.
There is also evidence for solvation over hydro-phobic groups on the protein surface. So-called hydrophobic hydration occurs because of the unfa-vorable nature of the interaction between water molecules and hydrophobic surfaces, resulting in the clustering of water molecules. Since this clustering is energetically unfavorable, such hydrophobic hydra-tion does not contribute to the protein stability. However, this hydrophobic hydration facilitates hy-drophobic interaction. This unfavorable hydration is diminished as the various hydrophobic groups come in contact either intramolecularly or intermolecularly, leading to the folding of intrachain structures or to protein–protein interactions.
Both the loosely and strongly bound water molecules can have an important impact, not only on protein stability but also on protein function. For example, certain enzymes function in non-aqueous solvent provided that a small amount of water, just enough to cover the protein surface, is present. Bound water can modulate the dynamics of surface groups, and such dynamics may be critical for enzyme function. Dried enzymes are, in general, inactive and become active after they absorb 0.2 g water per g protein. This amount of water is only sufficient to cover surface polar groups, yet may give sufficient flexibility for function.
Evidence that water bound to protein molecules has a different property from bulk water can be demonstrated by the presence of non-freezable water. Thus, when a protein solution is cooled below –40LC, a fraction of water, ~0.3 g water/g protein, does notfreeze and can be detected by high resolution NMR. Several other techniques also detect a similar amount of bound water. This unfreezable water reflects the unique property of bound water that prevents it from adopting an ice structure.