Section 23.4.5.1. Crambin

Crambin is a plant-seed hydrophobic protein of unknown function. It contains 46 amino-acid residues and was reported to form crystals that diffract to 0.88 Å resolution (Teeter & Hendrickson, 1979). The crystal structure of crambin was determined to 0.945 Å resolution directly from anomalous scattering by the six sulfur atoms involved in three disulfide bonds (Hendrickson & Teeter, 1981). Crambin is an amphipathic molecule in that the hydrophilic components (including six charged groups) are segregated from a mainly hydrophobic surface.

A total of 64 water molecules and two ethanol molecules were located in the electron-density map, despite the fact that the structure was determined in 60% ethanol. The overwhelming number of water molecules compared to ethanol is consistent with the results of the multiple-solvent crystal structures experiments described above for elastase (Mattos & Ringe, 1996).

Most of the 64 water molecules found in crambin interact with polar side chains in the typical manner described previously. The unusual information about solvent structure offered by the crambin model is that the arrangement of water molecules around hydrophobic residues is similar to that observed for clathrate hydrate structures (Teeter, 1991). Pentagonal water rings are observed to cap the Cδ2 atom of Leu18 as well as the hydrophobic methylene groups of Arg17 (Teeter, 1984, 1991). The set of five connected water rings is shown in Fig. 23.4.5.1. This ring cluster extends toward the protein, forming heterocyclic rings that are described in detail in the original article (Teeter, 1984).

Figure 23.4.5.1| top | pdf | van der Waals surface diagram of the water pentagons A, C, D and E in crambin viewed in the negative a direction. Rings A, C and E form a cap around leucine 18. Hydrophobic atoms are shown as dark circles, and water oxygens are shown as light circles. The methyl group of leucine 18 can be seen through the C ring. Adjacent translationally related molecules are shaded. The van der Waals radii used for the protein C, N and O atoms are 1.7, 1.4 and 1.4 Å, respectively, and for water oxygen, 1.8 Å. The larger radius is used for the water oxygens because hydrogen atoms have been omitted. Reprinted with the permission of the author from Teeter (1984).

Although crambin provides the clearest example of pentagonal water rings on a hydrophobic protein surface, it is not the only one. Other high-resolution crystal structures (better than 1.4 Å), such as insulin and cytochrome c, have also revealed pentagonal rings, but never to the extent seen in crambin (Teeter, 1984). This is very likely to be a general mode of interaction between water and hydrophobic moieties, be it in inorganic, organic, or biological molecules. The fact that it is not observed in protein structures in general may be related to the lower resolution of most X-ray structures, where it is not possible to model the more disordered areas where these patterns are likely to be found.