Characterization and 2D NMR study of the stable [9-21, 15-27] 2 disulfide intermediate in the folding of the 3 disulfide trypsin inhibitor EETI II.
The three disulfide Ecballium elaterium trypsin inhibitor II (EETI II) reduction with dithiothreitol (DTT) and reoxidation of the fully reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2-19] bridge. The NMR study showed a very strong structural conservation as compared to the native EETI II, suggesting that the bridges are the [9-21] and [15-27] native ones. The differences occurred in sections 2-7 (containing the free cysteine 2 and the Arg 4-Ile 5 active site) and 19-21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9-21, 15-27] arrangement and resulted in a well-conserved (7-28) segment.
Le-Nguyen
D
D
CCIPE, Montpellier, France.
Heitz
A
A
Chiche
L
L
el Hajji
M
M
Castro
B
B
D000595:Amino Acid Sequence; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010940:Plant Proteins; D011487:Protein Conformation; D017510:Protein Folding; D017421:Sequence Analysis; D014361:Trypsin Inhibitors / Q000737:chemistry*
D004220:Disulfides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; C063950:trypsin inhibitor EETI II protein, plant; D003545:Cysteine; D004229:Dithiothreitol