AN ENGINEERED AMINO-TERMINAL DOMAIN OF YEAST PHOSPHOGLYCERATE KINASE WITH NATIVE-LIKE STRUCTURE

Previous studies have suggested that the carboxy-terminal peptide (res idues 401-415) and interdomain helix (residues 185-199) of yeast phosp hoglycerate kinase, a two-domain enzyme, play a role in the folding an d stability of the amino-terminal domain (residues 1-184). A deletion mutant has been created in which the carboxy-terminal peptide is attac hed to the amino-terminal domain (residues 1-184) plus interdomain hel ix (residues 185-199) through a flexible peptide linker, thus eliminat ing the carboxy-terminal domain entirely. CD, fluorescence, gel filtra tion, and NMR experiments indicated that, unlike versions described pr eviously, this isolated N-domain is soluble, monomeric, compactly fold ed, native-like in structure, and capable of binding the substrate 3-p hosphoglycerate with high affinity in a saturable manner. The midpoint of the guanidine-induced unfolding transition was the same as that of the native two-domain protein (C-m similar to 0.8 M). The free energy change associated with guanidine-induced unfolding was one-third that of the native enzyme, in agreement with previous studies that evaluat ed the intrinsic stability of the N-domain and the contribution of dom ain-domain interactions to the stability of PGK. These observations su ggest that the C-terminal peptide and interdomain helix are sufficient for maintaining a native-like fold of the N-domain in the absence of the C-domain.