Effects of domain dissection on the folding and stability of the 43 kDa protein PGK probed by NMR.
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.
Reed
Michelle A C
MA
Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Hounslow
Andrea M
AM
Sze
K H
KH
Barsukov
Igor G
IG
Hosszu
Laszlo L P
LL
Clarke
Anthony R
AR
Craven
C Jeremy
CJ
Waltho
Jonathan P
JP
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
D010735:Phosphoglycerate Kinase