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Abstract:
We have solved the 2.0-Å resolution crystal structures of four cavity-creating Ile/LeufAla
mutations in the hydrophobic core of barnase and compare and contrast the structural responses to mutation
with those found for LeufAla mutations in T4 lysozyme. First, there are rearrangements of structure of
barnase that cause the cavities to collapse partly, and there is an approximately linear relationship between
the changes in stability and the volume of the cavity similar to that found for the mutants of T4 lysozyme.
Second, although it is currently accepted that hydrophobic cavities formed on the mutation of large
hydrophobic side chains to smaller ones are not occupied by water molecules, we found a buried water
molecule in the crystal structure of the barnase mutant Ile76fAla. 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 hydrophobic cavities produced by similar mutations in different
proteins reveals that they all contain a proportion of polar atoms in their linings. The availability of such
polar sites has implications for understanding folding pathways because a solvated core is presumed present
in the transition state for folding and unfolding. Notably, the hydrogen bond between the cavity-water
and the carbonyl group of Phe7 is also a marked early feature of very recent molecular dynamics simulations
of barnase denaturation [Caflisch, A., & Karplus, M. (1995) J. Mol. Biol. 252, 672-708]. It is possible
that cavities engineered into the hydrophobic cores of other proteins may contain water molecules, even
though they cannot be detected by crystallographic analysis.
