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.

The ribosome regulates flavodoxin folding

“…In native apoflavodoxin from Anabaena PCC 7119 the cofactor-binding site is flexible and FMN binds preferentially to native apo-protein (82)(83)(84). Full release of cofactor happens upon unfolding of this 169-residue long-chain flavodoxin (85). Both urea-and GuHCl-dependent equilibrium folding of apoflavodoxin are two-state (83,85,86).…”

“…Full release of cofactor happens upon unfolding of this 169-residue long-chain flavodoxin (85). Both urea-and GuHCl-dependent equilibrium folding of apoflavodoxin are two-state (83,85,86). During kinetic folding an intermediate transiently accumulates, whose unfolding is rate-limiting (3).…”

“…Thermal unfolding of Anabaena apoflavodoxin at equilibrium follows a threestate mechanism in which an intermediate, with spectroscopic properties of a MG, populates (85,91,92). A low-resolution structure of this species has been inferred from Phi-value analysis (92).…”

“…A low-resolution structure of this species has been inferred from Phi-value analysis (92). Large parts of the intermediate seem to have native-like topology, with somewhat weakened native interactions, but a 40-residue region is unfolded (85,91,92). The thermal intermediate neither resembles the MG that accumulates at low pH, nor the transition state for productive folding, nor the MG fragment.…”

Estudo da estabilidade térmica da hemoglobina extracelular gigante de Glossoscolex paulistus (HbGp): efeitos do estado de oxidação do ferro do grupo heme, pH e presença de surfactante

“…São descritos na literatura [27][28][29][30] vários modelos e equações que permitem obter parâmetros termodinâmicos do processo de desnaturação de proteínas. Esses modelos tem por base a condição de equilíbrio entre os estados nativo (proteína enovelada) e desnaturado (proteína desenovelada) e como ele é afetado por perturbações externas, tais como variações de pH, temperatura, adição de agentes caotrópicos ou surfactantes.…”