Cooperative Stabilization of a Molten Globule Apoflavodoxin Fragment

Abstract

We have destabilized apoflavodoxin by site-specific excision of its C-terminal helix. The resulting flavodoxin fragment (Fld1-149) is compact and monomeric at pH 7.0, with spectroscopic properties of a molten globule and a low conformational stability. To study if Fld1-149 is cooperatively stabilized, we have measured the equilibrium urea unfolding by fluorescence, circular dichroism, and size-exclusion chromatography. The three techniques produced coincident unfolding curves. Furthermore, the thermal unfolding seems also to be cooperative as the same temperature of half-denaturation is obtained using fluorescence and circular dichroism. Fld1-149 displays cold denaturation. The equilibrium properties of Fld1-149 demonstrate that molten globules lacking well-defined tertiary interactions can still be cooperatively stabilized and that cooperatively may appear in protein conformations of very low stability. This suggests that protein folding intermediates, can, in principle, be cooperatively stabilized.

The Long and Short Flavodoxins

“…5A). For this mechanism, the equations of the relaxation rates ( 1 and 2 ) and reduced amplitudes (normalized between 0 and 1 as described previously (21) vodoxin Stability and in the Stability of the Equilibrium Intermediate-The conformational stability of the apoflavodoxins of several species has been investigated, and it seems to be generally low (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). As for the stability of the holoform (carrying the FMN cofactor), there have been divergent reports pointing to either a significant or a rather marginal stabilization imparted by the presence of the prosthetic group (13,18).…”

“…Given their key biological function and a series of practical facts (i.e. they were among the first proteins for which x-ray structures became available (3,4), their purification in the pre-recombinant era was relatively easy, and they are reasonably stable to handle), flavodoxins were soon found to be convenient models to investigate electron transfer and molecular recognition (1,2) and, more recently, protein stability (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and folding (18 -21). Based on molecular weight and sequence comparisons, they were divided in two families: short-chain and long-chain flavodoxins (the latter containing an extra ϳ20-residue segment, subsequently shown in Refs.…”

“…The conformational stability and/or folding kinetics of three short apoflavodoxins (two different strains of Desulfovibrio desulfuricans (13,20) and Desulfovibrio vulgaris (18)) and two long ones (Azotobacter vinelandii (14 -17) and Anabaena PCC 7119 (5)(6)(7)(8)(9)(10)(11)(12)21)) have been reported. The available data, which have not always been gathered under the same conditions for the different proteins, suggest (but do not prove) that the long flavodoxins could display a more complex equilibrium and folding behavior than the short ones.…”

The ‘Relevant’ Stability of Proteins with Equilibrium Intermediates

“…The point is that the energetics of NI equilibria are so poorly understood that it is not clear whether the strategies found to stabilise two-state proteins will work well for proteins with equilibrium intermediates. If the energetics of protein intermediates are close to those of denatured states, there is no problem; but if, energetically, intermediates are not very different from the native state [7], then we face a difficult problem, because anything that stabilises the native conformation will similarly stabilise the intermediate, and the 'relevant' stability will not change much. …”

The ribosome regulates flavodoxin folding

“…In an apoflavodoxin variant whose stability has been reduced by excision of the C-terminal helix, the resulting protein fragment adopts a molten globular conformation at pH 7 (89,90). Its far-UV CD spectrum resembles the one of full-length, molten globular apoflavodoxin at acidic pH (86).…”