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 cooperativity 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 Kd = 0.2 microM, 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 synthease supports the hypothesis that these two (beta alpha)8-barrel proteins have evolved from a common ancestor.
Eder
J
J
Abteilung für Biophysikalische Chemie, Universität Basel, Switzerland.
Kirschner
K
K
D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D015951:Factor Xa / Q000737:chemistry; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000737:chemistry*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry
D009097:Multienzyme Complexes; D010446:Peptide Fragments; C052909:phosphoribosylanthranilate isomerase-indoleglycerol-phosphate synthase; D013762:Tetrahydrofolate Dehydrogenase; D015951:Factor Xa; D007209:Indole-3-Glycerol-Phosphate Synthase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases