Conformational stability of P22 tailspike proteins carrying temperature-sensitive folding mutations

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Abstract
The thermostable tailspike endorhamnosidase of Salmonella phage P22 provides a model system for comparing the role of amino acid sequences in determining the intracellular folding pathway with their role in stabilizing the mature structural protein. Complete Raman band assignments are given here for the native form of the tailspike trimer in aqueous solution. Once correctly folded and assembled, the wild-type and two well-characterized mutant proteins, tsfIle258 .fwdarw. Leu and tsfGly323 .fwdarw. Asp, exhibit the same secondary structure in solution, consisting predominantly of .beta.-strand (56 .+-. 5%) and turns (17 .+-. 2%). Raman bands that are sensitive indicators fo hydrogen-bonding interactions of tyrosine (phenolic OH) and tryptophan (indole NH) are unchanged between 30 and 80.degree. C in both wild type and tsf mutants. Similarly, Raman bands that are sensitive to changes in the hydrophobic environment of nonpolar side chains exhibit no significant temperature dependence in wild type and tsf mutants. In contrast, these conformational features are greatly altered by chemical denaturation of the tailspike with lithium halide and guanidine hydrochloride. In the chemically denatured tailspike, the .beta.-strand structure is substantially converted to irregular or "random coil" conformation. These findings confirm conclusions from physiological studies that the three-dimensional strucures of the tsf mutants, once stabilized at permissive temperatures, are equivalent to the native structure of the wild type, and this structure is maintained at temperatures far above those that block the folding of the chain into the final native conformation. The mutant sites therefore represent positions in the amino acid sequence where the side chains make important contributions to the stability of folding intermediates but do not contribute significantly to the stabilization of the native structure.