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

Abstract

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, -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 PCK. 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.Keywords: autonomously folding domains; isolated domains; phosphoglycerate kinase; protein engineering; protein folding As early as 1973, it was proposed that protein domains represent stable folding units (Wetlaufer, 1973). Since then, a number of isolated domains have been designed, expressed, and characterized (Minard et al

Site‐directed mutagenesis of proline 204 in the ‘hinge’ region of yeast phosphoglycerate kinase

“…Large proteolytic fragments including all of the C‐terminal domain refold after denaturation in a similar manner to the native enzyme as assessed by CD and fluorescence spectroscopy [27]. Deletion of the C‐terminal residues (401–415) has been found to disrupt the structure of both domains, but more so the N‐terminus [28] and a yeast PGK mutant, consisting only of residues 1–199 connected by two glycine residues to residues 401–415, was found to be native‐like in structure [29]. This suggests 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.…”

Comparison of Proteolytic Susceptibility in Phosphoglycerate Kinases from Yeast and E. coli: Modulation of Conformational Ensembles Without Altering Structure or Stability

“…Autonomous folding of the isolated N and C-domains from yeast PGK Since the N and C-domains of yPGK are known to fold autonomously 16,17,21,25,26 it is possible that a partial unfolding event due to selective uncoupling between the N and C-domains of yPGK is responsible for the difference in proteolysis rates. To test this hypothesis, we investigated the autonomous folding properties of the individual domains in isolation.…”

“…Fragments corresponding to the previously studied N and C-domains from yPGK (residues 1-198 and 402-416 with a two glycine linker, 17 and residues 185-416, respectively) were subcloned, expressed and purified for biophysical studies. As expected, the individual domains show CD spectra indicative of folded proteins with cooperative denaturation and temperature melts (Figures 6 and 7).…”

“…The three-dimensional structure of yeast PGK (yPGK) is composed of two independently folding and stable domains, and in solution has been shown to populate intermediates in its folding and unfolding pathways. [14][15][16][17][18][19][20][21][22] Hence, yPGK readily samples partially folded high-energy conformations on an experimental time scale. Based on this characterization one would expect the homologous ecPGK to be readily cleaved by proteases.…”

Role of loops in the folding and stability of yeast phosphoglycerate kinase

“…1[25]). PGK from various sources has been studied by several independent groups as a folding model representative of multidomain proteins [26–31].…”