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 (Cm approximately 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.
Sherman
M A
MA
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Chen
Y
Y
Mas
M T
MT
D000817:Anilino Naphthalenesulfonates; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D009682:Magnetic Resonance Spectroscopy; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D010735:Phosphoglycerate Kinase