title: The denatured state under native conditions: a non-native-like collapsed state of N-PGK.
abstract: The guanidinium-denatured state of the N-domain of phosphoglycerate kinase (PGK) has been characterized using solution NMR. Rather than behaving as a homogenous ensemble of random coils, chemical shift changes for the majority of backbone amide resonances indicate that the denatured ensemble undergoes two definable equilibrium transitions upon titration with guanidinium, in addition to the major refolding event. (13)C and (15)N chemical shift changes indicate that both intermediary states have distinct helical character. At denaturant concentrations immediately above the mid-point of unfolding, size-exclusion chromatography shows N-PGK to have a compact, denatured form, suggesting that it forms a helical molten globule. Within this globule, the helices extend into some regions that become beta strands in the native state. This predisposition of the denatured state to extensive non-native-like conformation, illustrates that, rather than directing folding, conformational pre-organization in the denatured state can compete with the normal folding direction. The corresponding reduction in control of the direction of folding as proteins become larger, could thus constitute a restriction on the size of protein domains.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.
authors.forename: Michelle A C
authors.initials: MA
authors.lastname: Reed
authors.forename: Clare
authors.initials: C
authors.lastname: Jelinska
authors.forename: Karl
authors.initials: K
authors.lastname: Syson
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cliff
authors.forename: Andrew
authors.initials: A
authors.lastname: Splevins
authors.forename: Tooba
authors.initials: T
authors.lastname: Alizadeh
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hounslow
authors.forename: Rosemary A
authors.initials: RA
authors.lastname: Staniforth
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: D001411:Geobacillus stearothermophilus / Q000201:enzymology; D019906:Nuclear Magnetic Resonance, Biomolecular; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D010735:Phosphoglycerate Kinase