title: A comparison of the urea-induced unfolding of apoflavodoxin and flavodoxin from Desulfovibrio vulgaris.
abstract: The kinetics and thermodynamics of the urea-induced unfolding of flavodoxin and apoflavodoxin from Desulfovibrio vulgaris were investigated by measuring changes in flavin and protein fluorescence. The reaction of urea with flavodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s(-1) in 3 m urea in 25 mm sodium phosphate at 25 degrees C), and it results in the dissociation of FMN. The rate of unfolding of apoflavodoxin depends on the urea concentration, while the reaction with the holoprotein is independent of urea. The rates decrease in high salt with the greater effect occurring with apoprotein. The fluorescence changes fit two-state models for unfolding, but they do not exclude the possibility of intermediates. Calculation suggests that 21% and 30% of the amino-acid side chains become exposed to solvent during unfolding of flavodoxin and apoflavodoxin, respectively. The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength. This effect is larger with phosphate than with chloride, and with apoflavodoxin than with flavodoxin. In low salt the conformational stability of the holoprotein is greater than that of apoflavodoxin, but in high salt the relative stabilities are reversed. It is calculated that two ions are released during unfolding of the apoprotein. It is concluded that the urea-dependent unfolding of flavodoxin from D. vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that flavodoxin dissociates. Small changes in flavin fluorescence occur at low concentrations of urea and these may reflect binding of urea to the holoprotein.
authors.affiliation: Department of Biochemistry, University College Dublin, Belfield, Dublin, Ireland.
authors.forename: Brian O
authors.initials: BO
authors.lastname: Nuallain
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Mayhew
mesh_terms: D001059:Apoproteins / Q000737:chemistry*; D016969:Desulfovibrio vulgaris / Q000737:chemistry*; D005486:Flavin Mononucleotide / Q000737:chemistry; D005418:Flavodoxin / Q000737:chemistry*; D005453:Fluorescence; D011487:Protein Conformation; D017510:Protein Folding; D014508:Urea / Q000494:pharmacology*
chemical_list: D001059:Apoproteins; D005418:Flavodoxin; C026932:apoflavodoxin; D005486:Flavin Mononucleotide; D014508:Urea