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Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues.

ABSTRACT: The oxidative folding of the Amaranthus alpha-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.

SUBMITTER: Cemazar M

PROVIDER: S-EPMC156273 | biostudies-literature | 2003 May

REPOSITORIES: biostudies-literature

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Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues.

Cemazar Masa M Zahariev Sotir S Lopez Jakob J JJ Carugo Oliviero O Jones Jonathan A JA Hore P J PJ Pongor Sandor S

Proceedings of the National Academy of Sciences of the United States of America 20030430 10

The oxidative folding of the Amaranthus alpha-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 polariz ...[more]

PMID: 12724517

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Project description:Disulfide bridges in proteins are formed by the oxidation of pairs of cysteine residues. These cross-links play a critical role in stabilizing the 3D-structure of small disulfide rich polypeptides such as hormones and venom toxins. The arrangement of the multiple disulfide bonds directs the peptide fold into distinct structural motifs that have evolved for resistance against biochemical and physical insults. These structural scaffolds have, therefore, proven to be very attractive in bioengineering efforts to develop novel biologics with applications in health and agriculture. Structural characterization of small disulfide rich peptides (DRPs) presents unique challenges when using commonly applied biophysical methods. NMR is the most commonly used method for studying such molecules, where the relatively small size of these molecules results in highly precise structural ensembles defined by a large number of distance and dihedral angle restraints per amino acid. However, in NMR the sulfur atoms that are involved in three of the five dihedral angles in a disulfide bond cannot be readily measured. Given the central role of disulfide bonds in the structure of these molecules, it is unclear what the inherent resolution of such NMR structures is when using traditional NMR methods. Here, we use an extensive set of long-range residual dipolar couplings (RDCs) to assess the resolution of the NMR structure of a disulfide-rich peptide. We find that structures based primarily on NOEs, yield ensembles that are equivalent to a crystallographic resolution of 2-3 Å in resolution, and that incorporation of RDCs reduces this to ~1-1.5 Å resolution. At this resolution the sidechain of ordered amino acids can be defined accurately, allowing the geometry of the cysteine bridges to be better defined, and allowing for disulfide-bond connectivities to be determined with high confidence. The observed improvements in resolution when using RDCs is remarkable considering the small size of these peptides.

Dataset Information

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues.

Publications

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues.

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