An NMR view of the folding process of a CheY mutant at the residue level.
The folding of CheY mutant F14N/V83T was studied at 75 residues by NMR. Fluorescence, NMR, and sedimentation equilibrium studies at different urea and protein concentrations reveal that the urea-induced unfolding of this CheY mutant includes an on-pathway molten globule-like intermediate that can associate off-pathway. The populations of native and denatured forms have been quantified from a series of 15N-1H HSQC spectra recorded under increasing concentrations of urea. A thermodynamic analysis of these data provides a detailed picture of the mutant's unfolding at the residue level: (1) the transition from the native state to the molten globule-like intermediate is highly cooperative, and (2) the unfolding of this state is sequential and yields another intermediate showing a collapsed N-terminal domain and an unfolded C-terminal tail. This state presents a striking similarity to the kinetic transition state of the CheY folding pathway.
Garcia
Pascal
P
Instituto de Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
Serrano
Luis
L
Rico
Manuel
M
Bruix
Marta
M
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D000072236:Methyl-Accepting Chemotaxis Proteins; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins