The concept of self-consistent J coupling evaluation exploits redundant structure information inherent in large sets of 3J coupling constants. Application to the protein Desulfovibrio vulgaris flavodoxin demonstrates the simultaneous refinement of torsion-angle values and related Karplus coefficients. The experimental basis includes quantitative coupling constants related to the polypeptide backbone φ torsion originating from a variety of heteronuclear 2D and 3D NMR correlation experiments, totalling 124 3J(HN,Hα), 129 3J(HN,C'), 121 3J(HN,Cβ), 128 3J(C'i-1,Hαi), 121 3J(C'i-1,C'i), and 122 3J(C'i-1,Cβi). Without prior knowledge from either X-ray crystallography or NMR data, such as NOE distance constraints, accurate φ dihedral angles are specified for 122 non-glycine and non-proline residues out of a total of 147 amino acids. Different models of molecular internal mobility are considered. The Karplus coefficients obtained are applicable to the conformational analysis of φ torsions in other polypeptides.

Recombinant Desulfovibrio vulgaris flavodoxin was produced in Escherichia coli. A complete backbone NMR assignment for the two-electron reduced protein revealed significant changes of chemical shift values compared to the oxidized protein, in particular for the flavine mononucleotide (FMN)-binding site. A comparison of homo- and heteronuclear NOESY spectra for the two redox states led to the assumption that reduction is not accompanied by significant changes of the global fold of the protein. The backbone dynamics of both the oxidized and reduced forms of D. vulgaris flavodoxin were investigated using two-dimensional 15N-1H correlation NMR spectroscopy. T1, T2 and NOE data are obtained for 95% of the backbone amide groups in both redox states. These values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-4559, 4559-4570]. A comparison of the two redox states indicates that in the reduced species significantly more flexibility occurs in the two loop regions enclosing FMN. Also, a higher amplitude of local motion could be found for the N(3)H group of FMN bound to the reduced protein compared to the oxidized state.

No abstract

No abstract

No abstract