Characterizing protein folding intermediates.

To determine the pathway by which a protein folds up, it is necessary to characterize the structures of folding intermediates and also to place these intermediates in the correct order on the kinetic pathway of folding. Noncovalent folding reactions are fast: typically they occur in seconds or less for small single-domain proteins. On the other hand, hours are required to obtain detailed structural information about a protein in solution by two-dimensional NMR spectroscopy. For proteins whose structures are stabilized by disullide bonds formed during the folding process, this dilemma can be sidestepped by covalently blocking unreacted thiol groups after partial oxidation of the cysteine residues to form disulfides, and then isolating and characterizing individual disutide intermediates [ 1 ] . In doing this, it is necessary toI relate the covalent chemistry of disulfide formation, as successive disullide bonds are formed, to the noncovalent folding process that is stabilized by H-bonds, hydrophobic interactions, and other weak interactions.