Lebowitz J, Kar S, Braswell E, McPherson S, Richard D L
Department of Microbiology, University of Alabama at Birmingham 35294.
Protein Sci. 1994 Sep;3(9):1374-82. doi: 10.1002/pro.5560030903.
Structural and biochemical evidence strongly supports a heterodimeric (p66p51) active form for human immunodeficiency virus-1 reverse transcriptase (RT). Heterodimer stability was examined by sedimentation analysis as a function of temperature and ionic strength. Using NONLIN regression software, monomer-dimer-trimer and monomer-dimer-tetramer association models gave the best fit to the analytical ultracentrifuge sedimentation equilibrium data. The heterodimer is the predominant form of RT at 5 degrees C, with a dimerization Ka value of 5.2 x 10(5) M-1 for both models. Ka values of 2.1 x 10(5) and 3.8 x 10(5) M-1 were obtained for the respective association models at 20 degrees C. RT in 50 and 100 mM Tris, pH 7.0, completely dissociates at 37 degrees C and behaves as an ideal monomeric species. The dissociation of RT as a function of increasing temperature was also observed by measuring the decrease in sedimentation velocity (sw,20). If the stabilization of the heterodimer was due primarily to hydrophobic interactions we would anticipate an increase in the association from 21 degrees C to 37 degrees C. The opposite temperature dependence for the association of RT suggests that electrostatic and hydrogen bond interactions play an important role in stabilizing heterodimers. To examine the effect of ionic strength on p66p51 association we determined the changes in sw,20 as a function of NaCl concentration. There is a sharp decrease in sw,20 between 0.10 and 0.5 M NaCl, leading to apparent complete dissociation. The above results support a major role for electrostatic interactions in the stabilization of the RT heterodimer.
结构和生化证据有力地支持了人类免疫缺陷病毒1型逆转录酶（RT）的异二聚体（p66p51）活性形式。通过沉降分析研究了异二聚体稳定性与温度和离子强度的关系。使用非线性回归软件，单体 - 二聚体 - 三聚体和单体 - 二聚体 - 四聚体缔合模型对分析超速离心沉降平衡数据拟合最佳。在5摄氏度时，异二聚体是RT的主要形式，两种模型的二聚化Ka值均为5.2×10⁵ M⁻¹。在20摄氏度时，各自缔合模型的Ka值分别为2.1×10⁵和3.8×10⁵ M⁻¹。在50和100 mM Tris，pH 7.0条件下的RT在37摄氏度时完全解离，表现为理想的单体形式。通过测量沉降速度（sw,20）的降低也观察到RT随温度升高的解离情况。如果异二聚体的稳定主要归因于疏水相互作用，我们预计从21摄氏度到37摄氏度缔合会增加。RT缔合的相反温度依赖性表明静电和氢键相互作用在稳定异二聚体中起重要作用。为了研究离子强度对p66p51缔合的影响，我们确定了sw,20随NaCl浓度的变化。在0.10至0.5 M NaCl之间，sw,20急剧下降，导致明显完全解离。上述结果支持静电相互作用在RT异二聚体稳定中起主要作用。
