NMR characterization of a single-cysteine mutant of Escherichia coli thioredoxin and a covalent thioredoxin-peptide complex.
The mechanism of disulfide reduction by thioredoxin in the cell is thought to occur through the formation and subsequent destruction of a mixed-disulfide intermediate between thioredoxin and the substrate. In order to model the interaction, we have prepared a mutant of Escherichia coli thioredoxin where the second cysteine residue of the active site has been replaced by an alanine residue. A specific covalent complex has been prepared between the remaining cysteine residue and a short cysteine-containing peptide. This paper describes the preparation and characterization of the mutant protein both free and in the peptide complex.
Jeng
M F
MF
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Reymond
M T
MT
Tennant
L L
LL
Holmgren
A
A
Dyson
H J
HJ
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics
D013879:Thioredoxins; D003545:Cysteine