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Protein Science (2004), 13:1182-1196. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Alteration of the disulfide-coupled folding pathway of BPTI by circular permutation

Grzegorz Bulaj, Rachel E. Koehn and David P. Goldenberg

Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, USA

(RECEIVED December 10, 2003; FINAL REVISION February 16, 2004; ACCEPTED February 16, 2004)



Abstract

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.

Keywords: bovine pancreatic trypsin inhibitor; protein folding; circular permutation; effective concentration

Abbreviations: BPTI, bovine pancreatic trypsin inhibitor • cBPTI, a circular form of BPTI generated by forming a peptide bond between the natural termini • cpBPTI, circularly permuted BPTI.

Reprint requests to: David P. Goldenberg, Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840, USA; e-mail: goldenberg{at}biology.utah.edu; fax: (801) 581-2174.

The specific variants are designated cp16, cp27, cp41, and cp46, where the numbers indicate the new N-terminal residues; amino acid replacements are identified by the wild-type residue, using the standard one-letter abbreviations, the residue number, and the residue type in the mutant protein; disulfide bonds are identified by the numbers of the Cys residues, and disulfide-bonded folding intermediates are indicated by square brackets enclosing the designations of the disulfides they contain.

GSSG and GSH, the oxidized and reduced forms of glutathione, respectively; DTTSS and DTTSHSH, the oxidized and reduced forms of dithiothreitol, respectively; GuHCl, guanidinium chloride; Tris-HCl, tris(hydroxymethyl)-aminomethane hydrochloride; EDTA, ethylenediaminetetraacetic acid; HPLC, high-performance liquid chromatography.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03563704.


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