International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F. ch. 23.4, p. 629   | 1 | 2 |

Section 23.4.3.3. The effect of tertiary structure on protein–water interactions

C. Mattosa* and D. Ringeb

aDepartment of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Raleigh, NC 02795, USA, and  bRosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South St, Waltham, MA 02254, USA
Correspondence e-mail:  mattos@bchserver.bch.ncsu.edu

23.4.3.3. The effect of tertiary structure on protein–water interactions

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At the tertiary level, there is an interdependence between protein surface shape and the extent of water binding (Kuhn et al., 1992[link]). Kuhn et al. (1992)[link] studied the binding locations of 10 837 water molecules found in 56 high-resolution crystal structures using fractal atomic density and surface-accessibility algorithms. They found strong correlations between the positions of water molecules and protein surface shape and amino-acid residue type. A probe sphere with the radius of a water molecule revealed that, in general, protein surfaces exhibit convex groove areas and concave contact surfaces. Although grooves account for approximately one quarter of a given protein surface, they bind half the water molecules. Furthermore, only within grooves was hydration found to be dependent on residue type, with charged and polar residues as well as main-chain nitrogen and oxygen atoms exhibiting a greater degree of hydration than the non-polar residues. Outside the grooves, there was a low residue-independent hydration level, with no distinction between main-chain and side-chain atoms (Kuhn et al., 1992[link]). Levitt & Park (1993)[link] discuss the paradox between the experimental observation that water molecules are crystallographically observed primarily in crevices (Kuhn et al., 1992[link]) and the results from theoretical calculations that argue that surface tension should make crevice waters bind less strongly (Nicholls et al., 1991[link]).

While the majority of the crystallographically observed water molecules appear on the outer protein surface, the internal protein packing is not perfect, so that the three-dimensional fold usually results in a number of internal cavities that can accommodate buried water molecules. The first analysis of such cavities was based on a small set of 12 proteins for which the authors characterized such sites by their size and area, as well as by whether or not they were occupied by crystallographically observed water molecules (Rashin et al., 1986[link]). More recently, two methodologically distinct studies of intramolecular cavities used much larger databases to provide extensive and mutually consistent conclusions regarding the properties of these sites (Hubbard et al., 1994[link]; Williams et al., 1994[link]). Hubbard et al. (1994)[link] analysed 121 protein chains, with no two possessing a pairwise identity greater than 40%. This study is based on a systematic method of determining the shape as well as the size of the internal cavities and categorizes each cavity as either `solvated' (with crystallographically visible water molecules) or `empty' (with no crystallographically visible water molecules), noting the amino-acid-residue preferences in each type. Hydrogen-bonding patterns were also noted within the solvated sites. The second study (Williams et al., 1994[link]) selected 75 non-homologous monomeric proteins, solved at 2.5 Å resolution or better. Although the authors noted the general shape, size and location of cavities, the focus of this study was on the buried water molecules and the hydrogen-bonding patterns that they form within these sites.

In general, larger proteins are able to tolerate larger cavity sizes than small proteins, and nearly all proteins with more than 100 amino-acid residues are found to have at least one cavity. These cavities are found in the protein interior at a variety of distances from the surface and reflect the difficulty of perfect packing within the core. In the database of 121 proteins (Hubbard et al., 1994[link]), 265 cavities were found to be `solvated' and 383 were `empty'. The solvated cavities tend to be nearer to the protein surface than the empty cavities. Nearly 60% of the solvated cavities are occupied by a single water molecule and are of spherical shape. About 20% accommodate two water molecules, and 20% more are found to contain larger clusters (Williams et al., 1994[link]). These tend to have an elongated cigar shape. The cavity volume can be as large as 216 Å3 (an elastase cavity containing seven water molecules). The solvated cavities tend to be larger than the empty ones, with average volumes of 39.4 and 20.7 Å3, respectively (Hubbard et al., 1994[link]). The mean volume per water molecule in a cavity is 27 Å3, as compared to 30 Å3 in bulk water, suggesting that a water molecule is not favourably squeezed into a volume comparable to its own (11.5 Å3), but rather occupies similar volumes upon transfer from the bulk into the protein interior.

Solvated cavities differ from empty ones not only in location and size within the protein, but also in the constitution of the amino-acid residues lining the cavity and the secondary-structure elements that are nearby. While 50% of the total cavity molecular surface is provided by polar atoms in solvated cavities, this fraction reflects only 16% of the empty cavity surface. Polarity, not size, is the predominant factor in determining the solvation state of a cavity. Interestingly, solvated cavities have more surface area provided by coil residues than the empty cavities, often found to be lined by residues in secondary structure (Hubbard et al., 1994[link]).

There is on average one buried water molecule per 27 amino-acid residues, although there is great variation between individual proteins. These water molecules most commonly form at least three hydrogen bonds with protein atoms or other buried water molecules. Only 18% of buried water molecules make two or fewer polar contacts. Of all of the hydrogen bonds made by buried water molecules, 53% are to protein backbone atoms, 30% to protein side-chain atoms, 17% to other buried water molecules, and 3% make no visible polar contacts at all (Williams et al., 1994[link]).

The appearance of cavities in the protein core is a consequence of the optimal packing of the protein polypeptide chain as it folds into the native, functional state. Where these cavities expose polar atoms to the hydrophobic protein core, one or more buried water molecules effectively become part of the structure, serving to maintain the protein integrity by fulfilling the hydrogen-bonding potential of atoms which are more favourably solvated.

References

First citation Hubbard, S. J., Gross, K.-H. & Argos, P. (1994). Intramolecular cavities in globular proteins. Protein Eng. 7, 613–626.Google Scholar
First citationKuhn, L., Siani, M. A., Pique, M. E., Fisher, C. L., Getzoff, E. D. & Tainer, J. A. (1992). The interdependence of protein surface topography and bound water molecules revealed by surface accessibility and fractal density measures. J. Mol. Biol. 228, 13–22.Google Scholar
First citation Levitt, M. & Park, B. H. (1993). Water: now you see it, now you don't. Structure, 1, 223–226.Google Scholar
First citation Nicholls, A., Sharp, K. A. & Honig, B. (1991). Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296.Google Scholar
First citation Rashin, A. A., Iofin, M. & Honig, B. (1986). Internal cavities and buried waters in globular proteins. Biochemistry, 25, 3619–3625.Google Scholar
First citation Williams, M. A., Goodfellow, J. M. & Thornton, J. M. (1994). Buried waters and internal cavities in monomeric proteins. Protein Sci. 3, 1224–1235.Google Scholar








































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