Plasticity in the Oxidative Folding Pathway of the High Affinity Nerita Versicolor Carboxypeptidase Inhibitor (NvCI)
Esperante, Sebastián (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Bronsoms, Sílvia (Universitat Autònoma de Barcelona. Servei de Proteòmica i Biologia Estructural)
Covaleda Cortés, Giovanni (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Trejo, Sebastián A. (Universitat Autònoma de Barcelona. Servei de Proteòmica i Biologia Estructural)
Avilés, Francesc X. (Francesc Xavier) (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Ventura, Salvador (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)

Data:	2017
Resum:	Nerita Versicolor carboxypeptidase inhibitor (NvCI) is the strongest inhibitor reported so far for the M14A subfamily of carboxypeptidases. It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β sheet connected by three loops and stabilized by three disulfide bridges. Here we report the oxidative folding and reductive unfolding pathways of NvCI. Much debate has gone on whether protein conformational folding guides disulfide bond formation or instead they are disulfide bonds that favour the arrangement of local or global structural elements. We show here that for NvCI both possibilities apply. Under physiological conditions, this protein folds trough a funnelled pathway involving a network of kinetically connected native-like intermediates, all sharing the disulfide bond connecting the two β-strands. In contrast, under denaturing conditions, the folding of NvCI is under thermodynamic control and follows a "trial and error" mechanism, in which an initial quasi-stochastic population of intermediates rearrange their disulfide bonds to attain the stable native topology. Despite their striking mechanistic differences, the efficiency of both folding routes is similar. The present study illustrates thus a surprising plasticity in the folding of this extremely stable small disulfide-rich inhibitor and provides the basis for its redesign for biomedical applications.
Ajuts:	Ministerio de Economía y Competitividad BFU2013-44763-P Ministerio de Economía y Competitividad BIO2013-44973-R
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Amino Acid Sequence ; Animals ; Binding Sites ; Carboxypeptidases ; Cloning, Molecular ; Crystallography, X-Ray ; Disulfides ; Gastropoda ; Gene Expression ; Genetic Vectors ; Humans ; Kinetics ; Models, Molecular ; Oxidation-Reduction ; Pichia ; Protease Inhibitors ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Denaturation ; Protein Folding ; Protein Interaction Domains and Motifs ; Protein Stability ; Protein Structure, Tertiary ; Recombinant Proteins ; Substrate Specificity ; Thermodynamics
Publicat a:	Scientific reports, Vol. 7 (2017) , art. 5457, ISSN 2045-2322

DOI: 10.1038/s41598-017-05657-7
PMID: 28710462

16 p, 3.9 MB

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