Local breathing and global unfolding in hydrogen exchange of barnase and its relationship to protein folding pathways.
We have measured the rate constants for exchange of amide protons in 15N-labeled wild-type barnase and a disulfide mutant that is more stable by 2 kcal.mol-1. The relative rate constants for exchange for wild type and mutant should reflect the changes in the equilibrium constants for local or global unfolding. The values for regions whose structure has been shown to be unaffected by the mutation fall into three subsets: those that are essentially unaffected by the mutation and so presumably exchange by local breathing; those where the energies change by close to 2 kcal.mol-1 and so presumably require global unfolding for exchange; and intermediate values that probably reflect a mixture of local and global unfolding in wild-type barnase. Amide protons that require the full change in unfolding energy are predominantly in the beta-sheet, which forms early in folding, but also include two that are involved in tertiary interactions that are known not to be formed until late in the folding pathway. Exchange in the major helix, which is known to form early, is largely unaffected by mutation and so exchanges by local breathing. There is thus no direct relationship between hydrogen-exchange behavior and the protein folding pathway. However, experiments on mutants of varying stability may provide further evidence on the sequence of events in folding.
Clarke
J
J
Medical Research Council Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, United Kingdom.
Hounslow
A M
AM
Bycroft
M
M
Fersht
A R
AR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002151:Calorimetry; D003001:Cloning, Molecular; D004220:Disulfides; D004926:Escherichia coli; D006860:Hydrogen Bonding; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D004220:Disulfides; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease