Effects of domain dissection on the folding and stability of the 43 kDa protein PGK probed by NMR

Research output: Contribution to journalArticle

Authors

  • AM Hounslow
  • KH Sze
  • IG Barsukov
  • LLP Hosszu
  • AR Clarke
  • CJ Craven
  • JP Waltho

Colleges, School and Institutes

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.

Details

Original languageEnglish
Pages (from-to)1189-1201
Number of pages13
JournalJournal of Molecular Biology
Volume330
Publication statusPublished - 25 Jul 2003