Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues.
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.
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, Trieste, Italy.
Masa
M
Cemazar
Sotir
S
Zahariev
Jakob J
JJ
Lopez
Oliviero
O
Carugo
Jonathan A
JA
Jones
P J
PJ
Hore
Sandor
S
Pongor
D003545:Cysteine; D016208:Databases, Factual; D004220:Disulfides; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D004220:Disulfides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D000516:alpha-Amylases; D003545:Cysteine