Secondary structure of the C-terminal DNA-binding domain of the transcriptional activator NifA from Klebsiella pneumoniae: Spectroscopic analyses

The conformation of the C-terminal DNA-binding domain of the transcriptiona l activator NifA from Klebsiella pneumoniae has been probed by circular dic hroism (CD), Fourier-transformed infrared (FTIR), and nuclear magnetic reso nance (NMR) spectroscopy in combination. Secondary structure prediction sug gests that the C-terminal half of the domain contains three alpha-helices. The spectra show that the domain is folded in the absence of DNA and of the N-terminal and central domains of NifA. The three spectroscopic techniques suggest slightly different proportions of secondary structural elements bu t all suggest that it contains about 33% alpha-helix. These results are in agreement with a previous prediction suggesting that NifA contains a helix- turn-helix motif and with the amount of alpha-helix predicted. The environm ent of the aromatic residues was examined by CD and NMR spectroscopy, which suggest that one or both of the tryptophan residues are involved in the te rtiary structure of the protein but that the tyrosine residue in the helix- turn-helix motif is solvent exposed and so available to bind to DNA. The th ermal melting profiles and pH-dependent structural changes were also examin ed by CD spectroscopy. This technique indicates that at low pH there is an increase in the secondary structure and interactions contributing to the te rtiary structure. Many of the acidic residues are predicted to be on a sing le helix, before the helix-turn-helix motif, which may therefore be importa nt for maintaining the structure and function of the C-terminal peptide; al ternatively, the N-terminal half of the domain may become more folded at lo w pH. (C) 1999 Academic Press.