Mutational analysis of hydrogen bonding residues in the BPTI folding pathway

Nine BPTI variants with replacements that remove one or more hydrogen bonds from the native protein were constructed, and the folding pathways of thes e proteins were determined by isolating and identifying the disulfide-bonde d intermediates that accumulated during unfolding and refolding. The forwar d and reverse rate constants for the individual steps in the folding pathwa ys for each protein were measured, providing a detailed description of the energetic effects of the substitutions. The native forms of eight of the ni ne variants were measurably destabilized, by 1-7 kcal/mol (1 cal = 4.184 J) , with an average effect of 1.6 kcal/mol per hydrogen bond removed. The fol ding pathways for the variants were found to be similar to that previously described for the wild-type protein, with the kinetically preferred mechani sm involving intramolecular rearrangements of intermediates with two disulf ide bonds. Some of the substitutions, however, significantly destabilized t he major intermediates and broadened the distribution of species with one o r two disulfide bonds, thus identifying residues that play important roles in stabilizing the normal intermediates and defining specificity in the fol ding process. The kinetic data also suggest that one residue, Asn43, may pl ay a distinctive role in defining the BPTI folding mechanism. Replacement o f this residue with either Gly or Ala appeared to stabilize the major trans ition states for folding and unfolding. In the native protein, the side-cha in of Asn43 participates directly in the hydrogen bonding pattern of the ce ntral beta -sheet, and the kinetic behavior of the Asn43 variants suggests that the major energy barriers in folding and unfolding may be due in part to fine steric constraints imposed by this structural element, together wit h those imposed by the chemical transition states for thiol-disulfide excha nge. (C) 2001 Academic Press.