The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

Abstract

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).

Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants

“…T4L mutants L121A/L133A and L133G have known crystal structures (14,15); the engineered cavities are large (>170 Å 3 ) and bind nonpolar ligands such as benzene (14). Although a crystal structure has not been reported for W138A, it apparently does not bind benzene (14).…”

“…T4 lysozyme (T4L) was selected for study because of the large database of crystal structures of ligand-binding cavity mutations and the corresponding thermodynamic characterization from Matthews and coworkers (4,(12)(13)(14)(15). Remarkably, comparison of the WT and mutant structures showed very little difference or limited local relaxation near the cavity, although the mutations were strongly destabilizing (4, 15).…”

Structure-Based Engineering of Internal Cavities in Coiled-Coil Peptides,

“…Most notable is the crystallographic two-fold symmetry axis along the helix axis in the P4 1 32 spacegroup, which results in each four-helix bundle being generated from just two unique chains, rather than four. The Glu6-Arg1 interhelical salt bridge is not observed in most cases, possibly because the N-terminal Arg residue is often not resolvable.…”

“…enzyme specificities, creating novel binding sites, and studying protein stability (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), but despite the diverse roles of natural cavities in biology, high-resolution structural investigations of model cavities in the aqueous milieu are rare. We believe studying designed cavities within synthetic peptides may provide models for understanding natural binding sites as well as designing novel receptors or biocatalysts.…”

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

“…74 Similarly, cavity-forming mutations also destabilize native proteins. 82 The studies on variants of α 2 D illustrate that solvent-exposed residues at interfaces can play an important, even decisive, role in defining structure. In summary, it is important to stress that many interactions are critical for maintaining a large free energy gap, and that these determinants of conformational specificity are typically distributed throughout the protein.…”