An engineered amino‐terminal domain of yeast phosphoglycerate kinase with native‐like structure

Previous studies have suggested that the carboxy‐terminal peptide (residues 401–415) and interdomain helix (residues 185–199) of yeast phosphoglycerate kinase, a two‐domain enzyme, play a role in the folding and stability of the amino‐terminal domain (residues 1–184). A deletion mutant has been created in which the carboxy‐terminal peptide is attached to the amino‐terminal domain (residues 1–184) plus interdomain helix (residues 185–199) through a flexible peptide linker, thus eliminating the carboxy‐terminal domain entirely. CD, fluorescence, gel filtration, and NMR experiments indicated that, unlike versions described previously, this isolated N‐domain is soluble, monomeric, compactly folded, native‐like in structure, and capable of binding the substrate 3‐phosphoglycerate 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 ∼ 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 evaluated the intrinsic stability of the N‐domain and the contribution of domain‐domain interactions to the stability of PGK. These observations suggest 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.