Hydrogen exchange in chymotrypsin inhibitor 2 probed by mutagenesis.
Two-dimensional NMR spectroscopy has been used to monitor hydrogen-deuterium exchange in chymotrypsin inhibitor 2. Application of two independent tests has shown that at pH 5.3 to 6.8 and 33 to 37 degrees C, exchange occurs via an EX2 limit. Comparison of the exchange rates of a number of mutants of CI2 with those of wild-type identifies the pathway of exchange, whether by local breathing, global unfolding or a mixture of the two pathways. For a large number of residues, the exchange rates were unaffected by mutations which destabilized the protein by up to 1.9 kcal mol(-1), indicating that exchange is occurring through local fluctuations of the native state. A small number of residues were found for which the mutations had the same effect on the rate constants for exchange as on the equilibrium constant for unfolding, indicating that these residues exchange by global unfolding. These are residues that have the slowest exchange rates in the wild-type protein. We see no correspondence between these residues and residues involved in the nucleation site for the folding reaction identified by protein engineering studies. Rather, the exchange behaviour of CI2 is determined by the native structure: the most protected amide protons are located in regions of hydrogen bonding, specifically the C terminus of the alpha-helix and the centre of the beta-sheet. A number of the most slowly exchanging residues are in the hydrophobic core of the protein.
Neira
J L
JL
MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, University Chemical Laboratory, UK.
Itzhaki
L S
LS
Otzen
D E
DE
Davis
B
B
Fersht
A R
AR
D001665:Binding Sites; D004735:Energy Transfer; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature
D010455:Peptides; D010940:Plant Proteins; C093336:chymotrypsin inhibitor 2; D006859:Hydrogen