Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy

被引:118
|
作者
Deprez, E
Tauc, P
Leh, H
Mouscadet, JF
Auclair, C
Brochon, JC
机构
[1] Ecole Normale Super, UMR CNRS 8532, Lab Biotechnol & Pharmacol Genet Appl, F-94235 Cachan, France
[2] Inst Gustave Roussy, UMR CNRS 8532, Lab Physicochim & Pharmacol Macromol Biol, F-94805 Villejuif, France
关键词
D O I
10.1021/bi000397j
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
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.
引用
收藏
页码:9275 / 9284
页数:10
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