Abstract
The oxidative folding and reductive unfolding pathways of leech carboxypeptidase inhibitor (LCI; four disulfides) have been characterized in this work by structural and kinetic analysis of the acid-trapped folding intermediates. The oxidative folding of reduced and denatured LCI proceeds rapidly through a sequential flow of 1-, 2-, 3-, and 4-disulfide (scrambled) species to reach the native form. Folding intermediates of LCI comprise two predominant 3-disulfide species (designated as III-A and III-B) and a heterogeneous population of scrambled isomers that consecutively accumulate along the folding reaction. Our study reveals that forms III-A and III-B exclusively contain native disulfide bonds and correspond to stable and partially structured species that interconvert, reaching an equilibrium prior to the formation of the scrambled isomers. Given that these intermediates act as kinetic traps during the oxidative folding, their accumulation is prevented when they are destabilized, thus leading to a significant acceleration of the folding kinetics. III-A and III-B forms appear to have both native disulfides bonds and free thiols similarly protected from the solvent; major structural rearrangements through the formation of scrambled isomers are required to render native LCI. The reductive unfolding pathway of LCI undergoes an apparent all-or-none mechanism, although low amounts of intermediates III-A and III-B can be detected, suggesting differences in protection against reduction among the disulfide bonds. The characterization of III-A and III-B forms shows that the former intermediate structurally and functionally resembles native LCI, whereas the III-B form bears more resemblance to scrambled isomers.
Highlights
The new view of protein folding, which has emerged in the recent years from a combination of experimental work and theoretical approximations, postulates the folding process as a parallel flow of molecules that follow multiple folding routes to reach the native state [1, 2]
Application of the oxidative folding and acid-trapping method has allowed the elucidation of folding pathways of several 3-disulfide proteins such as hirudin [15, 16], potato carboxypeptidase inhibitor (PCI) [17, 18], tick anticoagulant peptide (TAP) [19, 20], epidermal growth factor [21, 22], insulin-like growth factor (IGF-1) [23, 24], and the extensively investigated model of bovine pancreatic trypsin inhibitor (BPTI) (6, 7, 9 –11)
We have recently described both, the unfolding pathway and thermodynamic stability [37], and the oxidative folding process of this protein [38], showing that 3- and 4-disulfide intermediates act as kinetic traps along its folding pathway
Summary
Materials—Recombinant LCI was obtained by heterologous expression in Escherichia coli with an added glycine at the N terminus. Folding intermediates of LCI were trapped in a time course manner at selected times by mixing aliquots of the sample with 2% trifluoroacetic acid. Stop/Go Folding—Acid-trapped intermediates were isolated by RPHPLC, freeze-dried, and allowed to carry on the folding by dissolving the sample (0.5 mg/ml) in Tris-HCl buffer (0.1 M, pH 8.4), both in the absence and presence of 0.25 mM 2-mercaptoethanol. Reductive Unfolding—Native LCI and the 3-disulfide intermediates (0.5 mg) were dissolved in 1 ml of Tris-HCl buffer (0.1 M, pH 8.4) with different concentrations of DTT (0.1–100 mM). To monitor the kinetics of unfolding, time course aliquots of the samples were trapped with 2% trifluoroacetic acid, and analyzed by RP-HPLC. Disulfide Pairing Analysis of the Major Intermediates in the LCI Reductive Unfolding—The acid-trapped intermediates were purified by RP-HPLC and freeze-dried. The protein concentration of the LCI intermediates was determined from the A280 of the solution (LCI extinction coefficient: E0.1% ϭ 2.12)
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