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Plasticity and steric strain in a parallel beta-helix: Rational mutations in P22 tailspike protein
(2000)
By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect thermal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel beta-helix. We modified tailspike by rational single amino acid replacements at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) mutations in a similar structural context as two of the four known su mutations, which have been suggested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the beta-helix core. The high-resolution crystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the surface of the protein unaffected. This supports the notion that changes in the stability of an intermediate, in which the beta-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational 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 drastic 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 exemplifies the view arising from the statistical analysis of the distribution of backbone dihedral angles in known three-dimensional protein structures that the adoption of straight phi/psi angles other than the most favorable ones is often caused by side-chain interactions.
To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Forster theory, with polyproline treated as a rigid rod. At donor-acceptor distances much less than the Forster radius R-o, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R-0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Forster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor-acceptor distances
Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of CspTm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein (not, vert, similar104 s;1). Experiments on the unfolded protein without the added C- terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule Foerster resonance energy transfer (FRET) experiments.
To determine whether Förster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Förster theory, with polyproline treated as a rigid rod. At donor–acceptor distances much less than the Förster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Förster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor–acceptor distances.