The role of the cofactors in folding of Desulfovibrio desulfuricans flavodoxin and desulfoferrodoxin (DFX).

摘要

In this thesis, the roles of the cofactors for folding and stability of flavodoxin and desulfoferredoxin proteins from Desulfovibrio desulfuricans , a sulfate-reducing bacterium has been addressed.; Flavodoxins are proteins with an α/β topology that non-covalently coordinate a flavin mononucleotide (FMN) cofactor. These proteins from two strains of Desulfovibrio desulfuricans, ATCC 27774 and ATCC 29577 have been studied. The two proteins have 75.3% sequence identity, with the FMN-binding site being fully conserved. In order to characterize the folding pathway, three spectroscopic techniques were used: Far UV-CD, to characterize the secondary structure changes, fluorescence, to monitor the environment around the aromatic residues and visible absorption to monitor the FMN environment. The equilibrium-unfolding mechanism of Desulfovibrio desulfuricans flavodoxin (ATCC 27774) involves a native-like intermediate. In contrast, the equilibrium-unfolding mechanism of Desulfovibrio desulfuricans flavodoxin (ATCC 29557) is two-state. FMN stays bound to the unfolded polypeptide in both cases (using GuHCI as the denaturant). Holo-flavodoxin is only somewhat more stable than apo-flavodoxin. For Desulfovibrio desulfuricans flavodoxin (ATCC 29577), the folding kinetics were also probed. The holo-protein exhibits two-state kinetic behavior, albeit an additional slower phase is present during folding at very low denaturant concentrations. The extrapolated folding time in water for holo-flavodoxin, ∼280 μs (pH 7.0, 20°C), is in good agreement with that predicted from the protein's native-state topology. Desulfovibrio desulfuricans apoflavodoxin (ATCC 29577) on the other hand, exhibits bi-phasic folding and unfolding kinetics. The extrapolated folding times in water for both phases are slower than for holo-flavodoxin. The apo-protein data can be explained by an essentially off-pathway kinetic intermediate.; Desulfoferredoxin (Dfx) is a homodimer with the monomers linked through β-strand interactions in two domains. Each domain contains an iron center: Fe-(S-Cys) ₄ center in domain I and Fe-[S-Cys + (N-His)₄] center in domain 1I. Unfolding of Dfx was monitored by fluorescence, visible absorption and far-UV CD. Equilibrium unfolding of Dfx involves a monomeric intermediate with native-like secondary structure. Only after polypeptide unfolds do the iron ions dissociate. It seems that the iron centers, the amino-acid composition and to a lesser extent, the dimeric structure are factors governing Dfx's high thermodynamic stability.