Studies on protein stability with T4 lysozyme.

A series of mutants of phage T4 lysozyme have been constructed and have permitted a systematic analysis of different aspects of protein stability and folding. One striking result has been the ability of the protein to accommodate changes at many sites, yet still fold and retain activity. This shows that many amino acids in the sequence of the protein are nonessential for protein folding and stability. Such amino acids appear, predominantly, to be those that are mobile and/or largely exposed to solvent. Even though buried residues seem to be more important, it is still possible to change these as well. In some cases individual amino acids have been replaced. In other cases it has been found that a combination of substitutions permits repacking of the core. Such repacking is associated with adjustments of both the main chain and side chains. Only rarely do side chains rotate into radically new orientations. Taken together, substitutions of core residues confirm the overall importance of the hydrophobic effect as the dominant factor in stabilizing the folded structures of proteins. "Cavity-creating" substitutions of the form Leu-->Ala show, as expected, that the burial of the bulky leucine side chain within the core of the protein confers greater hydrophobic stabilization than is the case for the smaller alanine side chain. Leu-->Ala substitutions that create large cavities are especially destabilizing because they result in a loss of both hydrophobic and van der Waals interactions. In cases where the protein relaxes to reduce the size of the putative cavity, alternative van der Waals interactions are generated and the overall destabilization of the protein may be less severe than in cases where a large cavity is formed.