title: An evaluation of the use of hydrogen exchange at equilibrium to probe intermediates on the protein folding pathway.
abstract: BACKGROUND
  Methods have been developed recently for probing local fluctuations of protein structure using H/2H-exchange of amide protons at equilibrium. It has been suggested that equilibrium exchange methods can identify the order of events in folding pathways and detect folding cores. We have applied the procedure of measuring the effects of denaturant on the H/2H-exchange of amide protons of barnase, the folding pathway of which is well established.
  
  
  RESULTS
  The addition of relatively low concentrations of denaturant causes the mechanism of exchange of amide protons of barnase to change from EX2 to EX1 for the residues that require global unfolding for exchange to occur. This change of mechanism, which would have been missed by some of the standard tests, causes artefacts that could be easily misinterpreted. We also present the thermodynamic argument that measurements at equilibrium cannot give the order of events in folding.
  
  
  CONCLUSIONS
  Measurement of H/2H-exchange of amide protons at equilibrium, when applied correctly, is an excellent method for analyzing the equilibrium distribution of unfolded and partly folded states. It cannot, in theory and in practice, be used for determining protein folding pathways by itself.
authors.affiliation: Centre for Protein Engineering, MRC Centre, Cambridge, UK.
authors.forename: J
authors.initials: J
authors.lastname: Clarke
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000577:Amides / Q000737:chemistry; D001426:Bacterial Proteins; D003903:Deuterium; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D015394:Molecular Structure; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D012260:Ribonucleases / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D000577:Amides; D001426:Bacterial Proteins; D011506:Proteins; D011522:Protons; D006859:Hydrogen; D003903:Deuterium; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease