Knots in rings. The circular knotted protein Momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate.
The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys1-Cys18 disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.
Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane 4072 QLD, Australia.
Masa
M
Cemazar
Norelle L
NL
Daly
Sara
S
Häggblad
Kai Pong
KP
Lo
Ernie
E
Yulyaningsih
David J
DJ
Craik
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D047169:Cyclotides / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D003903:Deuterium; D004220:Disulfides / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry
D047169:Cyclotides; D004220:Disulfides; C409924:MCoTI-III protein, Momordica cochinchinensis; D018832:Molecular Chaperones; D010940:Plant Proteins; D014361:Trypsin Inhibitors; C091930:kalata B1; D006859:Hydrogen; D003903:Deuterium; D014357:Trypsin; D003545:Cysteine; D010100:Oxygen