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

被引:43
作者
Galán, A
Sot, B
Llorca, O
Carrascosa, JL
Valpuesta, JM
Muga, A [1 ]
机构
[1] Univ Basque Country, Consejo Super Invest Cientificas, Unidad Biofis, E-48080 Bilbao, Spain
[2] Univ Basque Country, Dept Bioquim & Biol Mol, E-48080 Bilbao, Spain
[3] Univ Autonoma Madrid, CSIC, Ctr Nacl Biotecnol, E-28049 Madrid, Spain
关键词
D O I
10.1074/jbc.M006861200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have studied the effect of macromolecular crowding reagents, such as polysaccharides and bovine serum albumin, on the refolding of tetradecameric GroEL from urea-denatured protein monomers, The results show that productive refolding and assembly strongly depends on the presence of nucleotides (ATP or ADP) and background macromolecules. Nucleotides are required to generate an assembly-competent monomeric conformation, suggesting that proper folding of the equatorial domain of the protein subunits into a native-like structure is essential for productive assembly. Crowding modulates GroEL oligomerization in two different ways. First, it increases the tendency of refolded, monomeric GroEL to undergo self-association at equilibrium. Second, crowding can modify the relative rates of the two competing self-association reactions, namely, productive assembly into a native tetradecameric structure and unproductive aggregation, This kinetic effect is most likely exerted by modifications of the diffusion coefficient of the refolded monomers, which in turn determine the conformational properties of the interacting subunits, If they are allowed to become assembly-competent before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place. Our data demonstrate that the spontaneous refolding and assembly of homo-oligomeric proteins, such as GroEL, can occur efficiently (70%) under crowding conditions similar to those expected in vivo.
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收藏
页码:957 / 964
页数:8
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