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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To determine the constraints on interactions within the core of a folded protein, we have analyzed the binding of 91 different compounds to an internal cavity created in the interior of phage T4 lysozyme by site-directed mutagenesis [Eriksson et al. (1992a) Nature 355, 371-373]. The cavity is able to accommodate a variety of small, mainly nonpolar, ligands. Molecules which do not appear to bind include those that are very polar, those that are too large, and those that have appropriate volume and polarity but inappropriate shape. Calorimetric analysis of 16 of these ligands reveals that their free energies of binding are poorly correlated with their solvent-transfer free energies. In addition, their enthalpies of binding are much larger than expected on the basis of transfer of the ligands from an aqueous to a nonpolar liquid phase. The binding energetics were analyzed by dividing the reaction into three processes: desolvation, immobilization, and packing. This analysis indicates that all three processes contribute to binding specificity. For a subset of these ligands that are structurally related, however, packing interactions in the protein interior are well modeled by the interactions of the ligands with octanol.

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts 5 0.3 A); in other cases mainchain atoms moved up to 2 A.In the case of Ile 29 -+ Ala the structure col!apsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 4 Ala (V149A) and Met 6 -+ Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the. cavity. The Val 149 -+ Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 -+ Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu + Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol" k 3 . Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu + Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).

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.

An overview is presented of some of the major insights that have come from studies of the structure, stability, and folding of T4 phage lysozyme. A major purpose of this review is to provide the reader with a complete tabulation of all of the variants that have been characterized, including melting temperatures, crystallographic data, Protein Data Bank access codes, and references to the original literature. The greatest increase in melting temperature (T m ) for any point mutant is 5.1°C for the mutant Ser 117 fi Val. This is achieved in part not only by hydrophobic stabilization but also by eliminating an unusually short hydrogen bond of 2.48 Å that apparently has an unfavorable van der Waals contact. Increases in T m of more than 3-4°C for point mutants are rare, whereas several different types of destabilizing substitutions decrease T m by 20°C or thereabouts. The energetic cost of cavity creation and its relation to the hydrophobic effect, derived from early studies of ''large-tosmall'' mutants in the core of T4 lysozyme, has recently been strongly supported by related studies of the intrinsic membrane protein bacteriorhodopsin. The L99A cavity in the C-terminal domain of the protein, which readily binds benzene and many other ligands, has been the subject of extensive study. Crystallographic evidence, together with recent NMR analysis, suggest that these ligands are admitted by a conformational change involving Helix F and its neighbors. A total of 43 nonisomorphous crystal forms of different monomeric lysozyme mutants were obtained plus three more for synthetically-engineered dimers. Among the 43 space groups, P2 1 2 1 2 1 and P2 1 were observed most frequently, consistent with the prediction of Wukovitz and Yeates.

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