The Unfolding Pathway of Leech Carboxypeptidase Inhibitor

Silvia Salamanca,Virtudes Villegas,Josep Vendrell,Li Li,Francesc X Aviles,Jui-Yoa Chang

doi:10.1074/jbc.m200040200

Abstract

The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable.

Highlights

Leech carboxypeptidase inhibitor (LCI)1 is the first metallocarboxypeptidase inhibitor found in leeches [1]
The denaturation curve that plots the fraction of native leech carboxypeptidase inhibitor (LCI) converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 M, respectively
We have recently described a new methodology to determine stability toward denaturants and to elucidate the unfolding pathway of disulfide-containing proteins [7, 8]. This approach is based on the observation that the presence of trace amounts of a thiol initiator during unfolding by denaturants generates a mixture of scrambled species mostly consisting of non-native disulfides that still maintain the native number of disulfide bonds

Summary

The Unfolding Pathway of Leech Carboxypeptidase Inhibitor*

We have recently described a new methodology to determine stability toward denaturants and to elucidate the unfolding pathway of disulfide-containing proteins [7, 8] This approach is based on the observation that the presence of trace amounts of a thiol initiator during unfolding by denaturants generates a mixture of scrambled species mostly consisting of non-native disulfides that still maintain the native number of disulfide bonds. This method has been applied to characterize the unfolding pathway of several disulfide-containing proteins, namely tick anticoagulant peptide (TAP) (3 disulfides) [8], PCI (3 disulfides) [9], and insulin-growth factor (IGF-1) (3 disulfides) [10]. The comparison to other disulfide-containing proteins offers an insight into the role of disulfide bonds in guiding the folding pathway and stabilizing the native fold

EXPERIMENTAL PROCEDURES

RESULTS

Unfolding of LCI

DISCUSSION

Sequence position

RNase A