STABLE SUBSTRUCTURES OF EIGHTFOLD BETA-ALPHA-BARREL PROTEINS - FRAGMENT COMPLEMENTATION OF PHOSPHORIBOSYLANTHRANILATE ISOMERASE

The (beta-alpha)8 (or "TIM")-barrel protein phosphoribosylanthranilate isomerase from Saccharomyces cerevisiae was cleaved between the sixth and seventh beta-alpha-module to test the capacity of the resulting fragments to adopt native format autonomously. The fragments, which were expressed from separate coding sequences, were soluble and monomeric. The amino-terminal fragment p1 was compact, possessed an almost nativelike far-UV but a strongly reduced near-UV CD spectrum, and unfolded cooperatively with guanidinium chloride. In contrast, the carboxyl-terminal fragment p2 was less compact than fragment p1, possessed only a weak far-UV and no detectable near-UV CD spectrum, and unfolded noncooperatively. The fragments assembled stoichiometrically to a complex with K(d) = 0.2-mu-M, which was enzymically almost fully active. The rate of assembly was limited by a first-order process, probably the isomerization of the carboxyl-terminal fragment p2 to an assembly-competent structure. These results support a folding mechanism that comprises an intermediate with the first six beta-alpha-units folded in roughly native format and the last two beta-alpha-units partially unfolded. The similar behavior of the analogous fragments of the alpha-subunit of tryptophan synthase supports the hypothesis that these two (beta-alpha)8-barrel proteins have evolved from a common ancestor.