The unfolding pathway of leech carboxypeptidase inhibitor.
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.
Salamanca
Silvia
S
Institut de Biotecnologia i Biomedicina i Departament de Bioquimica i Biologia Molecular, Universitat Autònoma de Barcelona 08193 Bellaterra, Spain.
Villegas
Virtudes
V
Vendrell
Josep
J
Li
Li
L
Aviles
Francesc X
FX
Chang
Jui-Yoa
JY
D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides / Q000032:analysis; D019791:Guanidine; D006146:Guanidines; D017879:Isothiocyanates; D007865:Leeches; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014508:Urea
D004220:Disulfides; D006146:Guanidines; D017879:Isothiocyanates; C116332:LCI protein, Hirudo medicinalis; D011506:Proteins; C054435:guanidine isothiocyanate; D014508:Urea; D019791:Guanidine