Excluded volume effects on the refolding and assembly of an oligomeric protein -: GroEL, a case study
Excluded volume effects on the refolding and assembly of an oligomeric protein -: GroEL, a case study
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DOI：
10.1074/jbc.m006861200
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发表时间：
2001-01-12
影响因子：
4.8
通讯作者：
Muga, A
中科院分区：
文献类型：
--
作者：
Galán, A;Sot, B;Muga, A
本文研究了大分子拥挤试剂如多糖和牛血清白蛋白对尿素变性蛋白质单体的十四聚体GroEL的复性的影响。结果表明，高效率的复性和组装强烈依赖于核苷酸（ATP或ADP）和背景大分子的存在。需要核苷酸产生组装能力的单体构象，这表明蛋白质亚基的赤道结构域适当折叠成天然样结构对于生产性组装是必不可少的。拥挤以两种不同的方式调节GroEL寡聚化。首先，它增加了重折叠的单体GroEL在平衡时进行自缔合的趋势。其次，拥挤可以改变两个竞争性自缔合反应的相对速率，即，生产性组装成天然十四聚体结构和非生产性聚集。这种动力学效应最有可能通过改变重折叠单体的扩散系数来施加，这反过来又决定了相互作用亚基的构象性质。如果允许它们在自缔合之前变得有组装能力，发生生产性低聚;否则发生非生产性聚集。我们的数据表明，自发重折叠和组装的同源寡聚蛋白质，如GroEL，可以有效地发生（70%）拥挤的条件下，类似于那些预期在体内。
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.