Unusual effect of salts on the homodimeric structure of NADH oxidase from Thermus thermophilus in acidic pH

Marek Stupák, Gabriel Žoldák, Andrej Musatov, Mathias Sprinzl, Erik Sedlák

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

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 pKa values of the conformational transition to a less acidic pH. Distortion of the dominant α-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 → π* electronic transitions at 222 nm. The unusual CD spectra correlated qualitatively with the theoretically calculated CD spectra of short α-helical structures and/or twisted β-sheets. Differences between the experimentally obtained CD spectra and theoretical calculations (AGADIR) of the α-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.

Original languageEnglish (US)
Pages (from-to)129-137
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1764
Issue number1
DOIs
StatePublished - Jan 2006
Externally publishedYes

Keywords

  • Circular dichroism
  • Dimeric interface
  • Flavoprotein
  • Molten globule
  • NADH oxidase

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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