title: Folding of Desulfovibrio desulfuricans flavodoxin is accelerated by cofactor fly-casting.
abstract: Folding of cofactor-binding proteins involves ligand binding in addition to polypeptide folding. We here assess the kinetic folding/binding landscape for Desulfovibrio desulfuricans flavodoxin that coordinates an FMN cofactor. The apo-form folds in a two-step process involving a burst-phase intermediate. Studies on Tyr98Ala and Trp60Ala variants reveal that these aromatics-that stack with the FMN in the holo-form-are not participating in the apo-protein folding pathway. However, these residues are essential for FMN interactions with the unfolded protein during refolding of holo-flavodoxin. Unfolding of wild-type holo-flavodoxin is coupled to FMN dissociation whereas for Tyr98Ala and Trp60Ala holo-variants, FMN dissociates before polypeptide unfolding. Both variants refold as apo-proteins before FMN rebinds. In sharp contrast, refolding of unfolded wild-type holo-flavodoxin is over an order of magnitude faster than that of the apo-form, the pathway does not include a burst-phase intermediate, and the speed is independent of FMN excess ratio. These observations demonstrate that FMN binds rapidly to the unfolded polypeptide and guides folding straight to the native state. As this path to functional D. desulfuricans holo-flavodoxin is faster than if the cofactor binds to pre-folded apo-protein, this is one of few examples where molecular recognition via a "fly-casting" mechanism is kinetically favored.
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA. bkmurali@utmb.edu
authors.forename: B K
authors.initials: BK
authors.lastname: Muralidhara
authors.forename: Ramesh
authors.initials: R
authors.lastname: Rathinakumar
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D045285:Desulfovibrio desulfuricans / Q000737:chemistry* / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D015394:Molecular Structure; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D005418:Flavodoxin; D008024:Ligands; D014443:Tyrosine; D005486:Flavin Mononucleotide; D019791:Guanidine; D000409:Alanine