Mutational analysis of the BPTI folding pathway: II. 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 to leucine amino acid replacement, were measured. From this analysis, the contributions of the four phenylalanine and four tyrosine residues to the stabilities of the native protein and the disulfide-bonded folding intermediates were determined. While the substitutions were found to destabilize the native protein by 2 to 7 kcal/mol, they had significantly 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 suggest that stabilizing interactions contribute less to conformational stability in the context of a partially folded intermediate than in a fully folded native protein, perhaps because of decreased cooperativity among the individual interactions. The kinetic analysis also provides new information about the transition states associated with the slowest steps in folding and unfolding, supporting previous suggestions that these transition states are extensively unfolded. Although the substitutions caused large changes in the distribution of folding intermediates and in the rates of some steps in the folding pathway, the kinetically-preferred pathway for all of the variants involved intramolecular disulfide rearrangements, as observed previously for the wild-type protein. These results suggest that the predominance of the rearrangement mechanism reflects conformational constraints present relatively early in the folding pathway.
Zhang
J X
JX
Department of Biology, University of Utah, Salt Lake City 84112, USA.
Goldenberg
D P
DP
D000478:Alkylation; D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D003553:Cystine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000235:genetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D004220:Disulfides; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D003553:Cystine; D007611:Aprotinin; D007930:Leucine