Alteration of the disulfide-coupled folding pathway of BPTI by circular permutation.
The kinetics of disulfide-coupled folding and unfolding of four circularly permuted forms of bovine pancreatic trypsin inhibitor (BPTI) were studied and compared with previously published results for both wild-type BPTI and a cyclized form. Each of the permuted proteins was found to be less stable than either the wild-type or circular proteins, by 3-8 kcal/mole. These stability differences were used to estimate effective concentrations of the chain termini in the native proteins, which were 1 mM for the wild-type protein and 2.5 to 4000 M for the permuted forms. The circular permutations increased the rates of unfolding and caused a variety of effects on the kinetics of refolding. For two of the proteins, the rates of a direct disulfide-formation pathway were dramatically increased, making this process as fast or faster than the competing disulfide rearrangement mechanism that predominates in the folding of the wild-type protein. These two permutations break the covalent connectivity among the beta-strands of the native protein, and removal of these constraints appears to facilitate direct formation and reduction of nearby disulfides that are buried in the folded structure. The effects on folding kinetics and mechanism do not appear to be correlated with relative contact order, a measure of overall topological complexity. These observations are consistent with the results of other recent experimental and computational studies suggesting that circular permutation may generally influence folding mechanisms by favoring or disfavoring specific interactions that promote alternative pathways, rather than through effects on the overall topology of the native protein.
Bulaj
Grzegorz
G
Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, USA.
Koehn
Rachel E
RE
Goldenberg
David P
DP
D000818:Animals; D007611:Aprotinin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004586:Electrophoresis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D010456:Peptides, Cyclic; C046916:circular bovine pancreatic trypsin inhibitor; D007611:Aprotinin; D014357:Trypsin