STRUCTURAL AND ENERGETIC RESPONSES TO CAVITY-CREATING MUTATIONS IN HYDROPHOBIC CORES - OBSERVATION OF A BURIED WATER MOLECULE AND THE HYDROPHILIC NATURE OF SUCH HYDROPHOBIC CAVITIES

We have solved the 2.0-Angstrom resolution crystal structures of four cavity-creating Ile/Leu-->Ala mutations in the hydrophobic core of bar nase and compare and contrast the structural responses to mutation wit h those found for Leu-->Ala mutations in T4 lysozyme. First, there are rearrangements of structure of barnase that cause the cavities to col lapse partly, and there is an approximately linear relationship betwee n the changes in stability and the volume of the cavity similar to tha t found for the mutants of T4 lysozyme. Second, although it is current ly accepted that hydrophobic cavities formed on the mutation of large hydrophobic side chains to smaller ones are not occupied by water mole cules, we found a buried water molecule in the crystal structure of th e barnase mutant Ile76-->Ala. A single hydrogen bond is formed between the water molecule and a polar atom, the carbonyl oxygen of Phe7, in the hydrophobic cavity that is formed on mutation. A survey of hydroph obic cavities produced by similar mutations in different proteins reve als that they all contain a proportion of polar atoms in their linings . The availability of such polar sites has implications for understand ing folding pathways because a solvated core is presumed present in th e transition state for folding and unfolding. Notably, the hydrogen bo nd between the cavity-water and the carbonyl group of Phe7 is also a m arked early feature of very recent molecular dynamics simulations of b arnase denaturation [Caflisch, A., & Karplus, M. (1995) J. Mol. Biol. 252, 672-708]. It is possible that cavities engineered into the hydrop hobic cores of other proteins may contain water molecules, even though they cannot be detected by crystallographic analysis.