Journal of Biomolecular NMR

, Volume 3, Issue 2, pp 133–149

1H and 15N resonance assignments and solution secondary structure of oxidized Desulfovibrio vulgaris flavodoxin determined by heteronuclear three-dimensional NMR spectroscopy

  • Brian J. Stockman
  • Annica Euvrard
  • David A. Kloosterman
  • Terrence A. Scahill
  • Richard P. Swenson
Research Papers

Summary

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 3JNHα coupling constants and 1HN exchange data, indicate that the secondary structure consists of a five-stranded parallel β-sheet and four α-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.

Keywords

Structural biology Isotopic enrichment Protein-ligand interactions Cofactor 

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Copyright information

© ESCOM Science Publishers B.V. 1993

Authors and Affiliations

  • Brian J. Stockman
    • 1
  • Annica Euvrard
    • 1
  • David A. Kloosterman
    • 1
  • Terrence A. Scahill
    • 1
  • Richard P. Swenson
    • 2
  1. 1.Upjohn LaboratoriesThe Upjohn CompanyKalamazooUSA
  2. 2.Department of BiochemistryOhio State UniversityColumbusUSA
  3. 3.Department of ChemistryNorthwestern UniversityEvanstonUSA

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