Excluded volume effects on the refolding and assembly of an oligomeric protein - GroEL, a case study

We have studied the effect of macromolecular crowding reagents, such as pol ysaccharides and bovine serum albumin, on the refolding of tetradecameric G roEL from urea-denatured protein monomers, The results show that productive refolding and assembly strongly depends on the presence of nucleotides (AT P or ADP) and background macromolecules. Nucleotides are required to genera te an assembly-competent monomeric conformation, suggesting that proper fol ding of the equatorial domain of the protein subunits into a native-like st ructure is essential for productive assembly. Crowding modulates GroEL olig omerization in two different ways. First, it increases the tendency of refo lded, monomeric GroEL to undergo self-association at equilibrium. Second, c rowding can modify the relative rates of the two competing self-association reactions, namely, productive assembly into a native tetradecameric struct ure and unproductive aggregation, This kinetic effect is most likely exerte d by modifications of the diffusion coefficient of the refolded monomers, w hich in turn determine the conformational properties of the interacting sub units, If they are allowed to become assembly-competent before self-associa tion, productive oligomerization occurs; otherwise, unproductive aggregatio n takes place. Our data demonstrate that the spontaneous refolding and asse mbly of homo-oligomeric proteins, such as GroEL, can occur efficiently (70% ) under crowding conditions similar to those expected in vivo.