Plasticity and steric strain in a parallel beta-helix: Rational mutations in the p22 tailspike protein

By means of genetic screens, a great number of mutations that affect the fo lding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease fo lding yields at high temperature, but hardly affect thermal stability of th e native trimeric structure when assembled at low temperature. Global suppr essor (SU) mutations mitigate this phenotype, Virtually all of these mutati ons are located in the central domain of tailspike, a large parallel beta-h elix, We modified tailspike by rational single amino acid replacements at t hree sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-cha in volume of a core residue, and (2) mutations in a similar structural cont ext as two of the four known su mutations, which have been suggested to sta bilize folding intermediates and the native structure by the release of bac kbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of f olding yields, refolding kinetics and thermal denaturation kinetics in vitr o show that the tsf phenotype can indeed be produced rationally by increasi ng the volume of side chains in the beta-helix core. The high-resolution cr ystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the a rrangement of the hydrogen-bonded backbone and thus the surface of the prot ein unaffected. This supports the notion that changes in the stability of a n intermediate, in which the beta-helix domain is largely formed, are the e ssential mechanism by which tsf mutations affect tailspike folding. A ratio nal design of su mutants, on the other hand, appears to be more difficult, The exchange of two residues in the active site expected to lead to a drast ic release of steric strain neither enhanced the folding properties nor the stability of tailspike, Apparently, side-chain interactions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifi es the view arising from the statistical analysis of the distribution of ba ckbone dihedral angles in known three-dimensional protein structures that t he adoption of phi/psi angles other than the most favorable ones is often c aused by side-chain interactions. (C) 2000 Wiley-Liss, Inc.