Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy.
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 (Mg(2+) and Mn(2+)) have a clear dissociative effect on the high multimeric forms of integrase. In addition, we observed that Mg(2+) and Mn(2+) 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 Zn(2+) stimulates dimerization. Interestingly, this role of Zn(2+) in the multimerization process was evident only in the presence of Mg(2+) 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.
Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée (UMR-CNRS 8532), Ecole Normale Supérieure (ENS) de Cachan, Cachan Cedex, France.
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D002413:Cations, Divalent / Q000737:chemistry; D002793:Cholic Acids / Q000737:chemistry; D003198:Computer Simulation; D003902:Detergents / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods; D019427:HIV Integrase / Q000737:chemistry* / Q000302:isolation & purification; D015497:HIV-1 / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008956:Models, Chemical; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D019065:Virus Assembly; D015032:Zinc / Q000737:chemistry
D002413:Cations, Divalent; D002793:Cholic Acids; D003902:Detergents; D012996:Solutions; D008345:Manganese; D014364:Tryptophan; D019427:HIV Integrase; D008274:Magnesium; D015032:Zinc; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate