Divita G, Rittinger K, Restle T, Immendörfer U, Goody R S
Max-Planck-Institut für Medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.
Biochemistry. 1995 Dec 19;34(50):16337-46. doi: 10.1021/bi00050a014.
The dissociation of dimeric reverse transcriptase (RT) of the human immunodeficiency virus (HIV) types 1 and 2 has been investigated using acetonitrile as a dissociating agent. The equilibrium transitions were monitored by combining different approaches (fluorescence spectroscopy, polymerase activity assay, and size-exclusion HPLC). The dissociation of RT induced a complete loss of polymerase activity and a 25% increase of the intrinsic fluorescence. It is fully reversible, and the midpoints of the equilibrium transition curves are dependent on the concentration of the enzyme used, suggesting a two-state transition model for the dissociation of RT in which dimers are in equilibrium with folded monomers. For both RTs, the heterodimeric form is more stable against dissociating agents and different pH than the corresponding homodimeric form. Moreover, heterodimeric HIV-2 RT exhibits a higher stability than HIV-1 RT, with a free energy of dissociation of 12.1 kcal/mol at pH 6.5 and 25 degrees C, instead of 10 kcal/mol for HIV-1 RT. The binding of a primer/template induces a marked conformational change in both RTs, shown by the lower accessibility of the tryptophans to quenchers and the increase in tryptophan heterogeneity, and stabilized the dimeric form of both RTs (10-100-fold). The central role of hydrophobic interactions in dimer formation has been revealed by the 30% increase of exposure of the tryptophan cluster to quenchers upon dissociation of RT and the binding of 4 equiv of 1-anilino-8-naphthalenesulfonate to the dissociated enzymes.
利用乙腈作为解离剂，对1型和2型人类免疫缺陷病毒（HIV）的二聚体逆转录酶（RT）的解离进行了研究。通过结合不同方法（荧光光谱法、聚合酶活性测定和尺寸排阻高效液相色谱法）监测平衡转变。RT的解离导致聚合酶活性完全丧失，固有荧光增加25%。这是完全可逆的，平衡转变曲线的中点取决于所用酶的浓度，表明RT解离存在一个二态转变模型，其中二聚体与折叠单体处于平衡状态。对于两种RT，异二聚体形式比相应的同二聚体形式对解离剂和不同pH更稳定。此外，异二聚体HIV-2 RT比HIV-1 RT表现出更高的稳定性，在pH 6.5和25℃下解离自由能为12.1千卡/摩尔，而HIV-1 RT为10千卡/摩尔。引物/模板的结合在两种RT中均诱导了明显的构象变化，表现为色氨酸对猝灭剂的可及性降低和色氨酸异质性增加，并稳定了两种RT的二聚体形式（10 - 100倍）。RT解离时色氨酸簇对猝灭剂的暴露增加30%以及4当量的1-苯胺基-8-萘磺酸盐与解离酶的结合揭示了疏水相互作用在二聚体形成中的核心作用。
