TY - JOUR
T1 - Effects of domain dissection on the folding and stability of the 43 kDa protein PGK probed by NMR
AU - Thompson, Michelle
AU - Hounslow, AM
AU - Sze, KH
AU - Barsukov, IG
AU - Hosszu, LLP
AU - Clarke, AR
AU - Craven, CJ
AU - Waltho, JP
PY - 2003/7/25
Y1 - 2003/7/25
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0038351770&partnerID=8YFLogxK
U2 - 10.1016/S0022-2836(03)00625-9
DO - 10.1016/S0022-2836(03)00625-9
M3 - Article
C2 - 12860138
VL - 330
SP - 1189
EP - 1201
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
ER -