IS THE CONTINUITY OF THE DOMAINS REQUIRED FOR THE CORRECT FOLDING OF A 2-DOMAIN PROTEIN

The role of domains in protein folding has been widely studied and dis cussed. Nevertheless, it is not clear whether the continuity of the do mains in a protein is an essential requirement in determining the fold ing pathway. Previous studies have shown that the isolated structural domains of the two-domain monomeric enzyme, yeast phosphoglycerate kin ase (yPGK), are able to fold independently into a quasinative structur e, but they neither reassociate nor generate a functional enzyme [Mina rd, P., Hall, L., Betton, J. M., Missiakas, D., & Yon, J. M. (1989) Pr otein Eng. 3, 55-60; Fairbrother, W. J., Bowen, D., Hall, L., Williams , R. J. P. (1989) Eur-. J. Biochem. 184, 617-625; Missiakas, D., Betto n, J. M., Minard, P., & Yen, J. M. (1990) Biochemistry 29, 8683-8689]. In the present work, two circularly permuted variants of the yPGK gen e were constructed. The natural adjacent chain termini were directly c onnected and the new extremities were created within the N-domain (at residues 71 and 72) or the C-domain (at residues 291 and 292), respect ively. These two proteins were overexpressed and purified. They exhibi t 14% and 23% of the wild-type enzyme activity, respectively. The two mutants fold in a compact conformation with slight changes in the seco ndary and tertiary structure probably related to the presence of a het erogeneous population of molecules. The unfolding studies reveal a lar ge decrease in stability. From the present data it appears that, altho ugh the circular permutations induce some perturbations in the structu re and stability of the protein, the protein, the continuity of the do mains is not required for the protein to reach a nativelike and functi onal structure.