Six "cavity-creating" mutants, Leu46----Ala (L46A), L99A, L118A, L121A, L133A, and Phe153----Ala (F153A), were constructed within the hydrophobic core of phage T4 lysozyme. The substitutions decreased the stability of the protein at pH 3.0 by different amounts, ranging from 2.7 kilocalories per mole (kcal mol-1) for L46A and L121A to 5.0 kcal mol-1 for L99A. The double mutant L99A/F153A was also constructed and decreased in stability by 8.3 kcal mol-1. The x-ray structures of all of the variants were determined at high resolution. In every case, removal of the wild-type side chain allowed some of the surrounding atoms to move toward the vacated space but a cavity always remained, which ranged in volume from 24 cubic angstroms (A3) for L46A to 150 A3 for L99A. No solvent molecules were observed in any of these cavities. The destabilization of the mutant Leu----Ala proteins relative to wild type can be approximated by a constant term (approximately 2.0 kcal mol-1) plus a term that increases in proportion to the size of the cavity. The constant term is approximately equal to the transfer free energy of leucine relative to alanine as determined from partitioning between aqueous and organic solvents. The energy term that increases with the size of the cavity can be expressed either in terms of the cavity volume (24 to 33 cal mol-1 A-3) or in terms of the cavity surface area (20 cal mol-1 A-2). The results suggest how to reconcile a number of conflicting reports concerning the strength of the hydrophobic effect in proteins.

The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure-factor least-squares refinement at 2.0 A resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc-bound water, seven more water molecules are identified in the active site. Assuming that Glu-106 is deprotonated at pH 8.5, some of the hydrogen bond donor-acceptor relations in the active site can be assigned and are described here in detail. The O gamma 1 atom of Thr-199 donates its proton to the O epsilon 1 atom of Glu-106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.

The hydrophobic cores of proteins are generally well packed, with few cavities. Mutations in which a bulky buried residue such as leucine or phenylalanine is replaced with a small residue such as alanine can create cavities in the core of a protein (our unpublished results). The sizes and shapes of such cavities can vary substantially depending on factors such as local geometry, whether or not a cavity already exists at the site of substitution, and the degree to which the protein structure relaxes to occupy the space vacated by the substituted residue. We show by crystallographic and thermodynamic analysis that the cavity created by the replacement Leu 99----Ala in T4 lysozyme is large enough to bind benzene and that ligand binding increases the melting temperature of the protein by 6.0 degrees C at pH 3.0. Benzene does not, however, bind to the cavity created by the Phe 153----Ala replacement. The results show that cavities can be engineered in proteins and suggest that such cavities might be tailored to bind specific ligands. The binding of benzene at an internal site 7 A from the molecular surface also illustrates the dynamic nature of proteins, even in crystals.