MUTATIONAL ANALYSIS OF THE BPTI FOLDING PATHWAY .2. EFFECTS OF AROMATIC-]LEUCINE SUBSTITUTIONS ON FOLDING KINETICS AND THERMODYNAMICS

The rates of the individual steps in the disulfide-coupled folding and unfolding of eight BPTI variants, each containing a single aromatic t o leucine amino acid replacement, were measured. From this analysis, t he contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide-bonded fold ing intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they Bad signifi cantly smaller effects on the intermediates that represent the earlier stages of folding, even when the site of the substitution was located within the ordered regions of the intermediates. These results sugges t that stabilizing interactions contribute less to conformational stab ility in the context of a partially folded intermediate than in a full y folded native protein, perhaps because of decreased cooperativity am ong the individual interactions. The kinetic analysis also provides ne w information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that t hese transition states are extensively unfolded. Although the substitu tions caused large changes in the distribution of folding intermediate s and in the rates of some steps in the folding pathway, the kinetical ly-preferred pathway for all of the variants involved intramolecular d isulfide rearrangements, as observed previously for the wild-type prot ein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.