The folding of an enzyme. V. H/2H exchange-nuclear magnetic resonance studies on the folding pathway of barnase: complementarity to and agreement with protein engineering studies

J Mol Biol. 1992 Apr 5;224(3):837-45. doi: 10.1016/0022-2836(92)90565-2.


Two major methods are currently being used to characterize transient intermediates during protein folding at the level of individual residues. Nuclear magnetic resonance (n.m.r.) measurements on the protection of peptide NH hydrogens against exchange with solvent during refolding can provide information about secondary structure formation. Protein engineering and kinetics can provide direct information about intramolecular interactions of protein side-chains and indirect evidence on secondary structure. These procedures have provided the most complete pictures so far about protein folding intermediates. Both methods have been applied to the characterization of an intermediate in the refolding of barnase. Although the two methods give complementary information, there are some regions of the protein where the methods overlap well. We show that, with one possible exception that is obscure, n.m.r. and protein engineering give identical results for those interactions that can be analysed by both methods. This suggests that these are valid approaches for the study of protein folding intermediates in the case of barnase and that the combination of the methods is a powerful analytical procedure. Information provided by n.m.r. data that is complementary to the protein engineering experiments is: (1) early formation of the C terminus of helix2; (2) early formation of helix3; (3) early formation of several beta-turns (46-49, 101-104 in loop5); and (5) partial formation of loop5. Confirmatory evidence of protein engineering data on the intermediate is: (1) helix1 is complete from residues 10 to 18; (2) the interactions between all beta-strands are present; (3) part of loop2 is not formed; (4) part of loop3 is formed; and (5) some specific tertiary interactions are not made. For some interactions the protein engineering and H/2H exchange methods overlap directly. The information obtained for direct overlap is self consistent.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / genetics
  • Bacillus / enzymology
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Kinetics
  • Magnetic Resonance Spectroscopy*
  • Molecular Probes
  • Mutagenesis
  • Protein Conformation*
  • Protein Engineering*
  • Ribonucleases / chemistry*
  • Ribonucleases / genetics


  • Amino Acids
  • Bacterial Proteins
  • Molecular Probes
  • Ribonucleases
  • Bacillus amyloliquefaciens ribonuclease