Plasticity in the Oxidative Folding Pathway of the High Affinity Nerita Versicolor Carboxypeptidase Inhibitor (NvCI)

Abstract

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.

Revisiting the Formation of a Native Disulfide Bond: Consequences for Protein Regeneration and Beyond

“…All said, there may be more “malleability” in the folding trajectory that previously imagined [ 121 ]. The intracellular pathways, aided by chaperones such as PDI and peptidyl prolyl isomerases, may differ from reductionist-derived processes.…”

A Chemical Biology Approach to Probing the Folding Pathways of the Inhibitory Cystine Knot (ICK) Peptide ProTx-II

“…Two models for these pathways have been proposed: a "BPTI-like" pathway where all of the folding intermediates contain only the disulfide bonds found in the native structure; and the "hirudin-like" pathway, in which a large number of heterogeneous folding intermediates can form, dominated by intermediates containing non-native disulfide bonds that are rearranged to give the native connectivity (Arolas et al, 2006;Chang, 2011). For many protein architectures, it has been demonstrated that conformational folding drives disulfide bond formation (Welker et al, 2001;Kosuri et al, 2012;Qin et al, 2015;Lv et al, 2018), whereas other proteins adopt intermediate pathways between these two extremes, or can follow different pathways under different conditions (Chang, 2004;Esperante et al, 2017).…”

Total synthesis of μ-conotoxin lt5d

“…11,12 These conditions are widely adopted to mimic physiological conditions favouring the formation of thermodynamically stable folded proteins and cysteine-rich peptides in vitro. [13][14][15] Fig . 1 presents the HPLC chromatograms (C 18 ) of pure linear and folded lt5d.…”

“… 11,12 These conditions are widely adopted to mimic physiological conditions favouring the formation of thermodynamically stable folded proteins and cysteine-rich peptides in vitro . 13–15 …”