title: Unusual effect of salts on the homodimeric structure of NADH oxidase from Thermus thermophilus in acidic pH.
abstract: The unusual salt-dependent behavior of the homodimeric flavoenzyme NADH oxidase from Thermus thermophilus in acidic pH has been studied using circular dichroism (CD) and sedimentation velocity. The native-like secondary and quaternary structures in acidic low ionic strength conditions were significantly perturbed by the addition of salts. The peptide region of the CD spectra showed a major salt-induced conformational change in the protein secondary structure. Sedimentation velocity experiments showed dissociation of the homodimeric structure of NADH oxidase in the presence of salt (>1 M). The new acidic conformation of the protein was stabilized by high ionic strength as indicated by a salt-induced increase in the melting temperature of the protein, and by a shift in the apparent pK(a) values of the conformational transition to a less acidic pH. Distortion of the dominant alpha-helical signal was expressed as the disappearance of the parallel polarized Moffitt exciton band at 208 nm without an accompanying loss of amplitude of n-->pi* electronic transitions at 222 nm. The unusual CD spectra correlated qualitatively with the theoretically calculated CD spectra of short alpha-helical structures and/or twisted beta-sheets. Differences between the experimentally obtained CD spectra and theoretical calculations (AGADIR) of the alpha-helical content of NADH oxidase indicate a role for non-local interactions in the protein conformation at high ionic strength and low pH. These findings indicate the importance of the homodimeric interface and electrostatic interactions for maintaining the structural integrity of this thermophilic protein.
authors.affiliation: Department of Biochemistry, Faculty of Science, P. J. Safárik University, Moyzesova 11, 041 54 Kosice, Slovakia.
authors.forename: Marek
authors.initials: M
authors.lastname: Stupák
authors.forename: Gabriel
authors.initials: G
authors.lastname: Zoldák
authors.forename: Andrej
authors.initials: A
authors.lastname: Musatov
authors.forename: Mathias
authors.initials: M
authors.lastname: Sprinzl
authors.forename: Erik
authors.initials: E
authors.lastname: Sedlák
mesh_terms: D019281:Dimerization; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000737:chemistry*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D009994:Osmolar Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012965:Sodium Chloride; D013696:Temperature; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D009097:Multienzyme Complexes; D012965:Sodium Chloride; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases