The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL.
The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL.
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DOI：
10.1021/bi001822b
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发表时间：
2001-04
期刊：
影响因子：
2.9
通讯作者：
A. L. Smoot;M. Panda;Bill T. Brazil;A. Buckle;and Alan R. Fersht;P. Horowitz
中科院分区：
文献类型：
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作者：
A. L. Smoot;M. Panda;Bill T. Brazil;A. Buckle;and Alan R. Fersht;P. Horowitz
研究了 bis-ANS 与 GroEL 功能性顶端结构域片段或微型伴侣（残基 191-345）结合后的疏水暴露程度，并与 GroEL 四聚体的疏水暴露程度进行了比较。尽管共有 7 个双-ANS 分子与该微型伴侣协同结合，但大多数疏水位点是在一到两个探针分子初始结合后诱导的。从低 bis-ANS 浓度下的平衡和动力学研究来看，很明显，天然顶端结构域转化为疏水表面增加的中间构象。该中间体结合额外的双-ANS 分子。酪氨酸荧光检测到的变性表明 bis-ANS 可以破坏顶端结构域的稳定性。 (i) bis-ANS 滴定、(ii) 在存在和不存在 bis-ANS 的情况下的尿素变性研究和 (iii) 顶端域的内在酪氨酸荧光研究的结果与 bis-ANS 与中间状态紧密结合、与天然状态相对较弱、与变性状态结合很少的模型一致。结果表明，由于顶端结构域与中间结构域的连接所施加的限制以及四级结构对运动的抑制，在完整的 GroEL 寡聚物中未观察到在顶端结构域片段中看到的构象变化。
The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.