Mutational analysis of the BPTI folding pathway: I. Effects of aromatic-->leucine substitutions on the distribution of folding intermediates.
The roles of aromatic residues in determining the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) were analyzed mutationally by examining the distribution of disulfide-bonded intermediates that accumulated during the refolding of protein variants in which tyrosine or phenylalanine residues were individually replaced with leucine. The eight substitutions examined all caused significant changes in the intermediate distribution. In some cases, the major effect was to decrease the accumulation of intermediates containing two of the three disulfides found in the native protein, without affecting the distribution of earlier intermediates. Other substitutions, however, led to much more random distributions of the intermediates containing only one disulfide. These results indicate that the individual residues making up the hydrophobic core of the native protein make clearly distinguishable contributions to conformation and stability early in folding: The early distribution of intermediates does not appear to be determined by a general hydrophobic collapse. The effects of the substitutions were generally consistent with the structures of the major intermediates determined by NMR studies of analogs, confirming that the distribution of disulfide-bonded species is determined by stabilizing interactions within the ordered regions of the intermediates. The plasticity of the BPTI folding pathway implied by these results can be described using conformational funnels to illustrate the degree to which conformational entropy is lost at different stages in the folding of the wild-type and mutant proteins.
Zhang
J X
JX
Department of Biology, University of Utah, Salt Lake City 84112, USA.
Goldenberg
D P
DP
D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics; D007930:Leucine / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D014443:Tyrosine / Q000235:genetics
D004220:Disulfides; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D007611:Aprotinin; D007930:Leucine