A test of the "jigsaw puzzle" model for protein folding by multiple methionine substitutions within the core of T4 lysozyme.

Abstract

To test whether the structure of a protein is determined in a manner akin to the assembly of a jigsaw puzzle, up to 10 adjacent residues within the core of T4 lysozyme were replaced by methionine. Such variants are active and fold cooperatively with progressively reduced stability. The structure of a seven-methionine variant has been shown, crystallographically, to be similar to wild type and to maintain a well ordered core. The interaction between the core residues is, therefore, not strictly comparable with the precise spatial complementarity of the pieces of a jigsaw puzzle. Rather, a certain amount of give and take in forming the core structure is permitted. A simplified hydrophobic core sequence, imposed without genetic selection or computer-based design, is sufficient to retain native properties in a globular protein.The cores of globular proteins consist of buried, primarily hydrophobic, amino acids. Tight packing of the amino acid side chains (1) has led to the idea that the size and shape of the nonpolar amino acids within the core may constrain or define the overall protein fold (2, 3). This "jigsaw puzzle" model of protein folding was originally introduced by Crick (4) as a "knobs into holes" description of a-helix packing and has been elaborated by Chothia et al. (5), and by Alber and co-workers (6). Here the jigsaw puzzle model refers to shape complementarity (3), not to the pathway of folding (7). The model is supported by the observation that changes in the sizes and shapes of residues within the cores of proteins are usually destabilizing (8-10). Also in support of the model, the structures of a-helical coiled coils appear to be determined by the shapes of the buried side chains (6). In contrast with this view, it has been shown that alternative core sequences that lead to viable proteins could be selected by random mutagenesis for both A-repressor (11) and T4 lysozyme (12), among others (13,14). It is possible, however, that a limited number of combinations of amino acids are viable and that they are the ones identified by the mutagenic selection. Here we explore an approach in which there is no selection other than the sites of substitution. MATERIALS AND METHODSWe chose methionine as a generic core-replacement residue for a combination of reasons. First, a methionine side chain occupies roughly the same volume as the frequently observed core residues leucine, isoleucine, and phenylalanine. It is, however, more flexible and can more readily adapt to occupy whatever space might be available. In this sense methionine contrasts with the rigid, predetermined shape of a piece of a jigsaw puzzle. Methionine also occurs relatively infrequently in known proteins (15). Thus multiple methionine substitutions would be expected to substantially change the composition of the core. Finally, we wondered if the intro-

Stabilization of GroEL minichaperones by core and surface mutations

“…The side-chain of Leu309 in M1 is forced to move backwards to avoid steric clash. It has been suggested that the mutation Met 3 Leu at entirely buried sites can stabilize a protein through an increase in sidechain hydrophobicity and the relative advantage in entropy of ordering a less¯exible leucine sidechain in the folded structures (Gassner et al, 1996). In this study, Met233 3 Leu is the most stabilizing of all the mutants, possibly emphasizing the effects of increased hydrophobic packing and conformational entropy.…”

L2′ loop is critical for caspase‐7 active site formation

“…In T4 lysozyme, a 1.9 kcal/mol change in DDG corresponds to approximately a 5 decrease in T m . 26 Thus, the 14 decrease in T m of unbound P214A could reflect a DDG of greater than 4 kcal/mol.…”

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

“…For example, the apolar cores of several natural proteins have been replaced with a single type, or a random collection, of different sized apolar residues with retention of folding and/or activity. [72][73][74][75][76][77] The changes often result in a loss in stability, and also in an increase in the dynamic properties of the side chains. However, these proteins retained folded structures, suggesting that hydrophobic interactions within the protein core are not the sole contributors to native structure.…”

“…Experiments in which the cores of natural proteins are repacked with a designed or randomly selected collection of hydrophobic side chains have shown that such proteins often retain a native-like structure, indicating that a variety of combinations of side chains can give rise to native folds. [72][73][74][75][76][77] It is tempting to conclude from these studies that the packing of hydrophobic side chains is not an important determinant of conformational specificity, and that only the pattern of hydrophobic and hydrophilic residues actually matters. However, only a fraction of the sequence of a protein is normally varied in these studies, and stabilizing interfacial interactions remain constant.…”