Abstract：
The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES7) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES7 and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES7) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES7 and CMC-GroES7 are decreased with a half-time of (23 ± 2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK ∼ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure.
摘要：
在一些寡聚蛋白质中发现了亚基交换，暗示其寡聚结构的短期解离，需要对四级结构在寡聚蛋白质稳定性和功能中的作用有新的认识。这里我们展示了pH值的影响，蛋白质浓度，和尿素对GroES七聚体(GroES7)亚基交换效率的影响。将等摩尔量的野生型（WT）GroES7及其用碘乙酸（97-羧甲基半胱氨酸或CMC-GroES7）修饰的Ala97Cys突变体的混合物在各种条件下孵育，并在聚丙烯酰胺凝胶中进行等电聚焦（IEF）。对于每个样本，有八个考马斯染色的电泳带显示出不同的电荷，这些电荷来自不同数量的包含的突变亚基，每个都带有额外的负电荷。这些条带的强度用于分析蛋白质亚基交换。使用横向尿素梯度凝胶电泳(TUGGE)评估蛋白质稳定性。在pH8.0时，对应于WT-GroES7和CMC-GroES7的初始条带的强度以（23±2）分钟的半衰期降低。交换随着pH的降低而降低，并且在pH5.2时似乎受到强烈阻碍，这是由于具有pK〜6.3的基团的质子化作用，从而稳定了蛋白质的四级结构。pH增加引起的蛋白质四级结构的不稳定，蛋白质浓度降低，或尿素加速GroES亚基交换。这项研究可以可视化寡聚蛋白质中的亚基交换，并证实其与蛋白质四级结构的稳定性直接相关。
