title: FMN binding and unfolding of Desulfovibrio desulfuricans flavodoxin: "hidden" intermediates at low denaturant concentrations.
abstract: The flavin mononucleotide (FMN) cofactor in Desulfovibrio desulfuricans flavodoxin stays associated with the polypeptide upon guanidine hydrochloride (GuHCl) induced unfolding. Using isothermal titration calorimetry (ITC), we determined the affinity of FMN for the flavodoxin polypeptide as a function of both urea and GuHCl concentrations (pH 7, 25 degrees C). The FMN affinity for folded and GuHCl-unfolded flavodoxin differs 10-fold, which is in agreement with the difference in thermodynamic stability between the apo- and holo-forms. In contrast, the urea-unfolded protein does not interact with FMN and equilibrium unfolding of holo-flavodoxin in urea results in FMN dissociation prior to polypeptide unfolding. ANS-binding, near-UV circular dichroism (CD), acrylamide quenching and FMN-emission experiments reveal the presence of native-like intermediates, not detected by far-UV CD and aromatic fluorescence detection methods, in low concentrations of both denaturants. Time-resolved experiments show that FMN binding is fastest at GuHCl concentrations where the native-like intermediate species is populated.
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street, Houston, TX 77251, USA.
authors.forename: B K
authors.initials: BK
authors.lastname: Muralidhara
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D002942:Circular Dichroism; D045285:Desulfovibrio desulfuricans / Q000737:chemistry*; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
chemical_list: D005418:Flavodoxin; D005486:Flavin Mononucleotide; D014508:Urea; D019791:Guanidine