HOMONUCLEAR AND HETERONUCLEAR NMR-STUDIES OF OXIDIZED DESULFOVIBRIO-VULGARIS FLAVODOXIN - SEQUENTIAL ASSIGNMENTS AND IDENTIFICATION OF SECONDARY STRUCTURE ELEMENTS

Recombinant Desulfovibrio vulgaris flavodoxin (molecular mass 16.3 kDa) was produced in Escherichia coli. The oxidized protein has been investigated with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Sequence-specific assignment of all backbone and most of the side chain H-1 and N-15 resonances has been obtained. The secondary structure has been inferred from the pattern of sequential, medium-, and long-range NOEs, together with information about slowly exchanging amide hydrogens and H(N)-H(alpha) spin-spin coupling constants. In solution, flavodoxin consists of a five-stranded parallel beta-sheet and four alpha-helices. Residues 3-9, 32-36, 52-58, 87-96, and 123-128 are involved in the beta-sheet whereas the alpha-helical regions comprise residues 13-28, 69-76, 104-114, and 134-148. Several proton resonances of the bound flavin mononucleotide cofactor have been assigned. NOE contacts between the prosthetic group and the apoprotein have been detected.