Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy
Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy
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DOI：
10.1021/bi000397j
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发表时间：
2000-08-08
期刊：
影响因子：
2.9
通讯作者：
Brochon, JC
中科院分区：
文献类型：
--
作者：
Deprez, E;Tauc, P;Brochon, JC
使用色氨酸残基作为探针，通过时间分辨荧光各向异性研究 HIV-1 整合酶的自组装特性。通过模拟分析，我们表明，合适的光子计数可以准确测定 20-80 ns 范围内的长旋转相关时间，从而可以将单体、二聚体和四聚体与更高寡聚形式的整合酶区分开来。高于 100 ns 的相关时间的准确度太低，无法将八聚体与其他较大的物种区分开来。然后在已知影响活性的不同参数下，在溶液中研究了广泛使用的去污剂溶解的整合酶的寡聚状态。在微摩尔范围内，整合酶分别在 25 和 35 摄氏度下以高级多聚体形式存在，例如八聚体和/或聚集体以及明确的四聚体。然而，整合酶在催化活性浓度（亚微摩尔范围）下是单体的。去污剂（NP-40 和 CHAPS）和二价阳离子辅助因子（Mg2+ 和 Mn2+）对整合酶的高多聚体形式具有明显的解离作用。此外，我们观察到Mg2+和Mn2+对低聚物状态和单体构象都有不同的影响。这可以部分解释为什么这两种金属阳离子在体外催化活性方面并不等效。相反，添加 Zn2+ 会刺激二聚化。有趣的是，Zn2+ 在多聚过程中的这种作用仅在 Mg2+ 存在时才明显，而 Mg2+ 本身不会诱导低聚。最后，强烈建议纯化过程中去污剂的存在对整合酶的正确自组装起到负面作用。因此，随附的论文 [Leh, H., et al. (2000) Biochemistry 39, 9285-9294]显示无去污剂的整合酶制剂具有与去污剂溶解的酶不同的自组装和催化特性。
Self-assembly properties of HIV-1 integrase were investigated by time-resolved fluorescence anisotropy using tryptophanyl residues as a probe. From simulation analyses, we show that suitable photon counting leads to an accurate determination of long rotational correlation times in the range of 20-80 ns, permitting the distinction of the monomer, dimer, and tetramer from higher oligomeric forms of integrase. The accuracy of correlation times higher than 100 ns is too low to distinguish the octamer from other larger species. The oligomeric states of the widely used detergent-solubilized integrase were then studied in solution under varying parameters known to influence the activity. In the micromolar range, integrase exists as high-order multimers such as an octamer and/or aggregates and a well-defined tetramer, at 25 and 35 degrees C, respectively. However, integrase is monomeric at catalytically active concentrations (in the sub-micromolar range). Detergents (NP-40 and CHAPS) and divalent cation cofactors (Mg2+ and Mn2+) have a dear dissociative effect on the high multimeric forms of integrase. In addition, we observed that Mg2+ and Mn2+ have different effects on both the oligomeric state and the conformation of the monomer. This could explain in part why these two metal cations are not equivalent in terms of catalytic activity in vitro. In contrast, addition of Zn2+ stimulates dimerization. Interestingly, this role of Zn2+ in the multimerization process was evident only in the presence of Mg2+ which by itself does not induce oligomerization. Finally, it is highly suggested that the presence of detergent during the purification procedure plays a negative role in the proper self-assembly of integrase. Accordingly, the accompanying paper [Leh, H., et al. (2000) Biochemistry 39, 9285-9294] shows that a detergent-free integrase preparation has self-assembly and catalytic properties different from those of the detergent-solubilized enzyme.