Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3: use of 3D 15N-HSQC-(TOCSY-NOESY)-15N-HSQC.
Virtually complete sequence specific 1H and 15N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F1-decoupled ROESY-15N-HSQC and 3D 15N-HSQC-(TOCSY-NOESY)-15N-HSQC using a single uniformly 15N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-15N-HSQC and a homonuclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without 13C/15N double labelling. Chemical shifts, 3J(alpha H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.
Nordstrand
K
K
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Ponstingl
H
H
Holmgren
A
A
Otting
G
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D054477:Glutaredoxins; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010088:Oxidoreductases; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
D001426:Bacterial Proteins; D054477:Glutaredoxins; D009587:Nitrogen Isotopes; D011506:Proteins; D010088:Oxidoreductases