1H and 15N resonance assignments and solution secondary structure of oxidized Desulfovibrio vulgaris flavodoxin determined by heteronuclear three-dimensional NMR spectroscopy.
Sequence-specific 1H and 15N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized Desulfovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing 1H-15N heteronuclear three-dimensional NMR experiments on uniformly 15N-enriched protein, pH 6.5, at 300 K. Many of the side-chain resonances have also been assigned. Observed medium-and long-range NOEs, in combination with 3JNH alpha coupling constants and 1HN exchange data, indicate that the secondary structure consists of a five-stranded parallel beta-sheet and four alpha-helices, with a topology identical to that determined previously by X-ray crystallographic methods. One helix, which is distorted in the X-ray structure, is non-regular in solution as well. Several protein-flavin NOEs, which serve to dock the flavin ligand to its binding site, have also been identified. Based on fast-exchange into 2H2O, the 1HN3 proton of the isoalloxazine ring is solvent accessible and not strongly hydrogen-bonded in the flavin binding site, in contrast to what has been observed in several other flavodoxins. The resonance assignments presented here can form the basis for assigning single-site mutant flavodoxins and for correlating structural differences between wild-type and mutant flavodoxins with altered redox potentials.
Stockman
B J
BJ
Upjohn Laboratories, Upjohn Company, Kalamazoo, MI 49007.
Euvrard
A
A
Kloosterman
D A
DA
Scahill
T A
TA
Swenson
R P
RP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D016969:Desulfovibrio vulgaris / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry*; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012996:Solutions
D001426:Bacterial Proteins; D005418:Flavodoxin; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D012996:Solutions; D006859:Hydrogen