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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. Proteins 2000;39:89-101.
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