1H and 15N nuclear magnetic resonance assignments, secondary structure in solution, and solvent exchange properties of azurin from Alcaligenes denitrificans

Abstract

Complete sequential H-1 and N-15 resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) = 14 000, 129 residues) from Alcaligenes denitrificans have been obtained at pH 5.5 and 32-degrees-C using homo- and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Comparison of the resonance assignments for the backbone protons with those of Pseudomonas aeruginosa azurin, which is 68% homologous in its amino acid sequence and has a very similar three-dimensional structure, showed a high similarity in chemical shift positions. After adjustment for random coil contributions the mean difference in N H chemical shifts is 0.00 ppm (root mean square width = 0.30 ppm), whereas for C(alpha) protons the mean difference is 0.09 ppm (root mean square width = 0.23 ppm). Characteristic NOE connectivities and 3J(HNalpha) values were used to determine the secondary structure of azurin in solution. Two beta-sheets, one helix, and nine tight and four helical turns were identified, and some long-range NOE contacts were found that connect the helix with the beta-sheets. The secondary structure obtained is in agreement with the structure derived from X-ray diffraction data [Baker, E. N. (1988) J. Mol. Biol. 203, 1071-1095]. Studies of the hydration of the protein in the vicinity of the copper ligand residue His117 revealed that the solvent-exposed N(epsilon2)H of His117 is in slow exchange with the bulk solvent. However, no evidence was obtained for the presence of a long-lived water molecule at the position corresponding to a well-defined water molecule observed in the crystal structures of A. denitrificans and Ps. aeruginosa azurin.