A proton nuclear magnetic resonance assignment and secondary structure determination of recombinant human thioredoxin.
Two-dimensional 1H NMR spectroscopy has been applied to a structural analysis of the reduced form of a recombinant human thioredoxin, a ubiquitous dithiol oxidoreductase recently isolated from an immunocompetent lymphoblastoid cell line. The sequential assignment of the spectrum, including all proline residues, has been accomplished by using experiments to demonstrate through-bond and through-space connectivities. The secondary structure has been determined by a qualitative interpretation of nuclear Overhauser effects, NH exchange data, and 3JHN alpha coupling constants. The secondary structure was found to be similar to that of the X-ray structure of Escherichia coli thioredoxin, consisting of a mixed five-stranded beta-sheet surrounded by four alpha-helices. The assignment and structural characterization of human thioredoxin was facilitated by the increased resolution and sensitivity afforded by a magnetic field strength of 600 MHz and required the use of two temperatures and two pH conditions to resolve ambiguities caused by a duplication of resonances. This duplication, extending from Phe-41 to Val-59, and including Lys-3-Ile-5, Val-24, Val-25, Asn-39, and Ile-101-Glu-103, appears to be due to heterogeneity arising from the presence or absence of the N-terminal methionine.
Forman-Kay
J D
JD
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Clore
G M
GM
Driscoll
P C
PC
Wingfield
P
P
Richards
F M
FM
Gronenborn
A M
AM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins; D012984:Software; D013879:Thioredoxins / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins