title: Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.
abstract: As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.
authors.affiliation: Department of Biomolecular Sciences, Wageningen Agricultural University, The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Steensma
authors.forename: M J
authors.initials: MJ
authors.lastname: Nijman
authors.forename: Y J
authors.initials: YJ
authors.lastname: Bollen
authors.forename: P A
authors.initials: PA
authors.lastname: de Jager
authors.forename: W A
authors.initials: WA
authors.lastname: van den Berg
authors.forename: W M
authors.initials: WM
authors.lastname: van Dongen
authors.forename: C P
authors.initials: CP
authors.lastname: van Mierlo
mesh_terms: D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry*; D005420:Flavoproteins / Q000737:chemistry*; D006859:Hydrogen; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: D005418:Flavodoxin; D005420:Flavoproteins; D015335:Molecular Probes; C111991:holoflavodoxin II; D006859:Hydrogen