title: Effects of domain dissection on the folding and stability of the 43 kDa protein PGK probed by NMR.
abstract: The characterization of early folding intermediates is key to understanding the protein folding process. Previous studies of the N-domain of phosphoglycerate kinase (PGK) from Bacillus stearothermophilus combined equilibrium amide exchange data with a kinetic model derived from stopped-flow kinetics. Together, these implied the rapid formation of an intermediate with extensive native-like hydrogen bonding. However, there was an absence of protection in the region proximal to the C-domain in the intact protein. We now report data for the intact PGK molecule, which at 394 residues constitutes a major extension to the protein size for which such data can be acquired. The methods utilised to achieve the backbone assignment are described in detail, including a semi-automated protocol based on a simulated annealing Monte Carlo technique. A substantial increase in the stability of the contact region is observed, allowing protection to be inferred on both faces of the beta-sheet in the intermediate. Thus, the entire N-domain acts concertedly in the formation of the kinetic refolding intermediate rather than there existing a distinct local folding nucleus.
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Michelle A C
authors.initials: MA
authors.lastname: Reed
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hounslow
authors.forename: K H
authors.initials: KH
authors.lastname: Sze
authors.forename: Igor G
authors.initials: IG
authors.lastname: Barsukov
authors.forename: Laszlo L P
authors.initials: LL
authors.lastname: Hosszu
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Clarke
authors.forename: C Jeremy
authors.initials: CJ
authors.lastname: Craven
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
mesh_terms: D000465:Algorithms; D003198:Computer Simulation; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006860:Hydrogen Bonding; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009010:Monte Carlo Method; D010735:Phosphoglycerate Kinase / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D010735:Phosphoglycerate Kinase