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.

Enzymes are thought to use their ordered structures to facilitate catalysis. A corollary of this theory suggests that enzyme residues involved in function are not optimized for stability. We tested this hypothesis by mutating functionally important residues in the active site of T4 lysozyme. Six mutations at two catalytic residues, Glu-11 and Asp-20, abolished or reduced enzymatic activity but increased thermal stability by 0.7-1.7 kcal mol-1. Nine mutations at two substrate-binding residues, Ser-117 and Asn-132, increased stability by 1.2-2.0 kcal-mol'1, again at the cost of reduced activity. X-ray crystal structures show that the substituted residues complement regions of the protein surface that are used for substrate recognition in the native enzyme. In two of these structures the enzyme undergoes a general conformational change, similar to that seen in an enzyme-product complex. These results support a relationship between stability and function for T4 lysozyme. Other evidence suggests that the relationship is general.The ordered, functional structures of proteins reflect two tendencies that are often opposed. On one hand, proteins fold to minimize their free energy. On the other hand, they organize themselves to recognize a ligand or a transition state (1). Minimizing free energy leads to well-packed hydrophobic interiors and hydrophilic exteriors (2). Maximizing function leads to active-site clefts where charged and polar groups are sequestered from water (3, 4) and where hydrophobic patches are exposed to solvent.The hypothesis that there is a balance between stability and function can be stated most strongly as follows: protein residues that contribute to catalysis or ligand binding are not optimal for protein stability. This "stability-function" hypothesis predicts that it usually should be possible to replace residues known to be important for function, reducing protein activity but concomitantly increasing stability of the folded protein.Here we describe experiments that directly test the stabilityfunction hypothesis in T4 lysozyme, an enzyme well characterized for the effects of mutation on structure and stability (5). Five residues were replaced (Table 1 and Fig. 1). These included two residues implicated in chemical catalysis, 11,12), as well as thyee others thought to have a role in substrate binding, Gly-30, Ser-117, and Asp-132. We measured the thermodynamic stability and kinetic activity of the mutant lysozymes. To determine the structural consequences of the substitutions, we determined x-ray crystal structures for several of these proteins. MATERIALS AND METHODSMutagenesis and Protein Purification. Mutations were introduced into the T4 lysozyme gene borne by M13 phage derivative M13mpl8 T4e by mismatched oligonucleotides using the method of Kunkel et al (13) as detailed (6, 14).

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