Conformational stability of apoflavodoxin

Abstract

Flavodoxins are ar/p proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from A n a b~n a PCC 71 19 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 1 1 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 'H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a prornising system for the analysis of the stability and folding of cr/0 proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.

A differential scanning calorimetric study of the thermal unfolding of apo‐ and holo‐cytochrome b562

“…The enthalpy of unfolding of the apoprotein is about 50 kcal mol", a little less than half that of the holoprotein, and the heat capacity change for unfolding is substantially decreased, to about 0.3 kcal K" mol". These values are generally larger than have been observed in other systems for partially folded species or "molten globules" both in magnitude and as a fraction of the native protein values (Xie et al, 1991;Yutani et al, 1992;Ogasahara et al, 1993;Martinez et al, 1995;Genzor et al, 1996;Kreimer et al, 1996). Dramatic changes occur in the structure of cyt bs62 when the heme is removed (Fig.…”

How FMN Binds to Anabaena Apoflavodoxin

“…1 and Ref. 16), and in addition, an extensive characterization of the solution behavior of the two forms has been performed (7,(17)(18)(19)(20)(21)(22)(23)(24). The binding of FMN to apoflavodoxins from several species seems to occur in one step (5,25), although the molecular details are not known.…”

The ribosome regulates flavodoxin folding

“…Its far-UV CD spectrum resembles the one of full-length, molten globular apoflavodoxin at acidic pH (86). The structure of this MG is homogeneously weakened compared to the one of full-length native protein (90).…”

“…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).…”

“…This MG is structurally different from the two thermal intermediates identified at neutral pH (99,102). Whereas under acidic conditions the MG of H. pylori apoflavodoxin is soluble and monomeric (98), the MG of Anabaena apoflavodoxin aggregates massively (86). For H. pylori apoflavodoxin at neutral pH, the ensemble of molecules comprises native protein, the MG discovered by acid unfolding and the two intermediates revealed by thermal unfolding of the protein (99).…”