Section 22.2.5.5. Hydrogen bonds with water molecules

Water molecules, with their small size and double-donor, double-acceptor hydrogen-bonding capability, are ideal for completing intramolecular hydrogen-bonding networks, e.g. by linking two proton acceptor atoms, or two protein donor atoms, that cannot otherwise interact. Thus, buried water molecules, making multiple hydrogen bonds, help satisfy the hydrogen-bond potential of internal polar atoms and contribute to protein stability; internal waters average about three hydrogen bonds each (Baker & Hubbard, 1984; Williams et al., 1994). From the survey of Williams et al. (1994), most (58%) occupy discrete cavities, while 22% are in clusters housing two waters and 20% are in larger clusters; some examples of larger clusters are given in Baker & Hubbard (1984). Buried waters are often conserved between homologous proteins (Baker, 1995), and each buried water–protein hydrogen bond is estimated to stabilize a folded protein by, on average, 0.6 kcal mol⁻¹ (1 kcal mol⁻¹ = 4.184 kJ mol⁻¹) (Williams et al., 1994). More loosely bound external waters exchange much more rapidly and presumably contribute less energetically.

Several patterns of hydrogen bonding are consistently observed. Water molecules are most often seen interacting with oxygen atoms rather than nitrogen atoms and acting as hydrogen-bond donors rather than acceptors. Possible reasons include the greater number of acceptor sites in proteins and the fewer geometrical restrictions imposed by acceptors (Baker & Hubbard, 1984; Baker, 1995). There is also a predominance of interactions with main-chain atoms rather than side-chain atoms: on average ∼40% with main-chain C=O groups, 15% with main-chain NH and 45% with side-chain groups (Baker & Hubbard, 1984; Thanki et al., 1988). Favoured main-chain binding sites include the N- and C-termini of helices, C=O groups on the solvent-exposed sides of helices, the edge strands of β-sheets, and the ends of strands where they add extra inter-strand hydrogen bonds at the position where the strands diverge (Thanki et al., 1991). Among side chains, the most highly hydrated appear to be Asp and Glu, whose COO⁻ groups bind, on average, two water molecules each (Baker & Hubbard, 1984; Thanki et al., 1988). On the other hand, the best-ordered water sites are created by residues whose side chains simultaneously make hydrogen bonds to other protein atoms (His, Asp, Asn, Arg) or may be sterically restricted (Tyr, Trp).

The distributions of water molecules around protein groups follow the geometrical patterns expected from simple bonding ideas (Baker & Hubbard, 1984; Thanki et al., 1988). Interactions with NH groups are linear, and those with C=O groups show a preferred angle of ∼130° at the oxygen-atom acceptor, consistent with interaction with an oxygen-atom lone pair; restriction to the peptide plane is not very strong, however. Although the distributions around polar side chains generally follow the expected patterns (Thanki et al., 1988), there is little evidence of ordered water clusters around non-polar groups. This may be because water clusters need to be `anchored' by hydrogen bonding to polar groups to be seen crystallographically.