Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Abstract

The oxidative folding of the Amaranthus ␣-amylase inhibitor, a 32-residue cystine-knot protein with three disulfide bridges, was studied in vitro in terms of the disulfide content of the intermediate species. A nonnative vicinal disulfide bridge between cysteine residues 17 and 18 was found in three of five fully oxidized intermediates. One of these, the most abundant folding intermediate (MFI), was further analyzed by 1 H NMR spectroscopy and photochemically induced dynamic nuclear polarization, which revealed that it has a compact structure comprising slowly interconverting conformations in which some of the amino acid side chains are ordered. NMR pulsed-field gradient diffusion experiments confirmed that its hydrodynamic radius is indistinguishable from that of the native protein. Molecular modeling suggested that the eight-membered ring of the vicinal disulfide bridge in MFI may be located in a loop region very similar to those found in experimentally determined 3D structures of other proteins. We suggest that the structural constraints imposed on the folding intermediates by the nonnative disulfides, including the vicinal bridge, may play a role in directing the folding process by creating a compact fold and bringing the cysteine residues into close proximity, thus facilitating reshuffling to native disulfide bridges.T he cystine knot, or the knottin fold, is a compact structure consisting of a short triple-stranded antiparallel ␤-sheet reinforced by three disulfide bridges that form a topological knot [see Amaranthus ␣-amylase inhibitor (AAI) structure, Fig. 1A Inset]. It is found both in small peptides and as a domain of larger proteins (1) and in addition, it is a recurrent substructure of larger cysteine-rich motifs such as the well known conotoxin fold (2). Despite their small size, cystine knots can fulfill a large variety of functions ranging from ion-channel blocking to enzyme inhibition, which makes them ideal protein engineering scaffolds for industrial applications (3, 4). Because cystine knots occur in many unrelated species (fungi, plants, snails, and spiders, etc.), it is presumed that this fold has emerged via convergent evolution (2). The compactness and versatility of this fold are best illustrated by the fact that both the shortest peptide inhibitor of ␣-amylases, AAI [32 amino acids (Fig. 1 A)] (5-8) and the shortest lectin molecule known so far (9), are members of this family.Cystine knot peptides are known to form readily in vitro under the conditions of oxidative folding (4). In this work, we describe an analysis of the oxidative folding intermediates of AAI, which shows that the native protein (N) forms via several fully oxidized intermediates with nonnative disulfide bonds, including the vicinal disulfide 17-18. NMR spectroscopy revealed that the main folding intermediate has a structure as compact as the completely folded peptide, comprising a number of slowly interconverting backbone conformations. On the basis of the experimental data, we suggest that the compactness, a prerequisi...

Oxidative Folding of Amaranthus α-Amylase Inhibitor

“…AAI peptide was produced manually on a 1-mmol scale by solid-phase peptide synthesis using an Fmoc (N-(9-fluorenyl)methoxycarbonyl) methodology (33,39). The crude peptide was isolated with yield Ͼ90% and purified to homogeneity (Ͼ98%, RP-HPLC).…”

“…The crude peptide was isolated with yield Ͼ90% and purified to homogeneity (Ͼ98%, RP-HPLC). The procedure was essentially as described elsewhere (39), with the exception of cysteine residues, which were introduced as Fmoc-Cys(Trt)-OPfp (33,44).…”

“…Samples were eluted with linear gradients of solvents A (H 2 O, 0.1% trifluoroacetic acid) and B (CH 3 CN, 0.1% trifluoroacetic acid). The disulfide connectivities of intermediates were determined as described elsewhere (33). The detailed conditions of oxidative folding from native AAI and MFI, reductive unfolding from native AAI and MFI, and folding in the presence of guanidine hydrochloride (GdnHCl) and DsbC are all indicated in the corresponding figure legends.…”

“…The cystine-knot motif is known for the high structural stabilization of its protein scaffold that makes it suitable for molecular engineering and drug design (43). The oxidative folding of AAI has been characterized (33) to include many heterogeneous disulfide intermediate species along its folding pathway, and their disulfide structures have been determined. Apart from the native species (disulfide bonds: 1-18, 8 -23, and 17-31), five other three-disulfide species appear on the folding pathway, all of which contain non-native disulfide bonds (see Fig.…”

“…These intermediates (MFI, I-5, and I-6) appear to be the first folding intermediates observed to have a vicinal disulfide bond. MFI has been studied in detail for its tertiary structure and the solvent accessibility of aromatic residues Tyr 21 , Tyr 27 , Tyr 28 , and Trp 5 (33). Because of its "bead-like" disulfide structure (1-8, 17-18, 23-31) and the high contact order that it confers, MFI has been assigned a significant role in the folding process (33).…”