title: Native-specific stabilization of flavodoxin by the FMN cofactor: structural and thermodynamical explanation.
abstract: Flavodoxins are useful models to investigate protein/cofactor interactions. The binding energy of the apoflavodoxin-FMN complex is high and therefore the holoflavodoxin is expected to be more stable than the apoprotein. This expectation has been challenged by reports on the stability of Desulfovibrio desulfuricans flavodoxin indicating that FMN binds to the unfolded polypeptide with similar affinity as to the native state, thus causing no net effect on protein stability. In previous work, we have analyzed in detail the stability of the apoflavodoxin from Anabaena PCC 7119 and the energetics of its functional complex with FMN. Here, we use the Anabaena holoprotein to directly investigate the contribution of the bound cofactor to protein stability through a detailed analysis of the chemical and thermal denaturation equilibria. Our data clearly shows that FMN binding largely stabilizes the protein towards both chemical and thermal denaturation, and that the stabilization observed at 25 degrees C in low ionic strength conditions is precisely the one expected if full release of the cofactor takes place upon flavodoxin unfolding. On the other hand, the binding of FMN to the native polypeptide is shown to simplify the thermal unfolding so that, while apoflavodoxin follows a three-state mechanism, the holoprotein unfolds in a two-state fashion. Comparison of the X-ray structure of native apoflavodoxin with the phi-structure of the thermal intermediate indicates that the increase in cooperativity driven by the cofactor originates in its preferential binding to the native state, which is a consequence of the disorganization in the intermediate of the FMN binding loops and of an adjacent longer loop.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias & Biocomputation and Complex Systems Physics Institute, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: L A
authors.initials: LA
authors.lastname: Campos
authors.forename: J
authors.initials: J
authors.lastname: Sancho
mesh_terms: D000595:Amino Acid Sequence; D017033:Anabaena; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D005418:Flavodoxin; D005486:Flavin Mononucleotide