1H and15N resonance assignments and solution secondary structure of oxidizedDesulfovibrio vulgarisflavodoxin determined by heteronuclear three-dimensional NMR spectroscopy

✍ Scribed by Brian J. Stockman; Annica Euvrard; David A. Kloosterman; Terrence A. Scahill; Richard P. Swenson

• Publisher: Springer Netherlands
• Year: 1993
• Tongue: English
• Weight: 933 KB
• Volume: 3
• Category: Article
• ISSN: 0925-2738
• DOI: 10.1007/bf00178258

✦ Synopsis

Sequence-specific ~H and 15N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized DesulJovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing 1H-~SN 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 3Jt~Hc ~ coupling constants and IH N exchange data, indicate that the secondary structure consists of a five-stranded parallel ]3-sheet and four e~-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 2H20, 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.