Singh Sushmita, Zlotnick Adam
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
J Biol Chem. 2003 May 16;278(20):18249-55. doi: 10.1074/jbc.M211408200. Epub 2003 Mar 13.
For many protein multimers, association and dissociation reactions fail to reach the same end point; there is hysteresis preventing one and/or the other reaction from equilibrating. We have studied in vitro assembly of dimeric hepatitis B virus (HBV) capsid protein and dissociation of the resulting T = 4 icosahedral capsids. Empty HBV capsids composed of 120 capsid protein dimers were more resistant to dissociation by dilution or denaturants than anticipated from assembly experiments. Using intrinsic fluorescence, circular dichroism, and size exclusion chromatography, we showed that denaturants dissociate the HBV capsids without unfolding the capsid protein; unfolding of dimer only occurred at higher denaturant concentrations. The apparent energy of interaction between dimers measured in dissociation experiments was much stronger than when measured in assembly studies. Unlike assembly, capsid dissociation did not have the concentration dependence expected for a 120-subunit complex; consequently the apparent association energy systematically varied with reactant concentration. These data are evidence of hysteresis for HBV capsid dissociation. Simulations of capsid assembly and dissociation reactions recapitulate and provide an explanation for the observed behavior; these results are also applicable to oligomeric and multidomain proteins. In our calculations, we find that dissociation is impeded by temporally elevated concentrations of intermediates; this has the paradoxical effect of favoring re-assembly of those intermediates despite the global trend toward dissociation. Hysteresis masks all but the most dramatic decreases in contact energy. In contrast, assembly reactions rapidly approach equilibrium. These results provide the first rigorous explanation of how virus capsids can remain intact under extreme conditions but are still capable of "breathing." A biological implication of enhanced stability is that a triggering event may be required to initiate virus uncoating.
对于许多蛋白质多聚体而言，缔合和解离反应无法达到相同的终点；存在滞后现象，阻止其中一个和/或另一个反应达到平衡。我们研究了二聚体乙型肝炎病毒（HBV）衣壳蛋白的体外组装以及由此产生的T = 4二十面体衣壳的解离。由120个衣壳蛋白二聚体组成的空HBV衣壳对稀释或变性剂解离的抗性比组装实验预期的更强。利用内源荧光、圆二色性和尺寸排阻色谱，我们表明变性剂可使HBV衣壳解离而不会使衣壳蛋白展开；二聚体的展开仅在更高的变性剂浓度下才会发生。在解离实验中测得的二聚体之间的表观相互作用能比在组装研究中测得的要强得多。与组装不同，衣壳解离不具有120亚基复合物预期的浓度依赖性；因此，表观缔合能随反应物浓度系统地变化。这些数据证明了HBV衣壳解离存在滞后现象。衣壳组装和解离反应的模拟重现并解释了观察到的行为；这些结果也适用于寡聚和多结构域蛋白。在我们的计算中，我们发现解离受到中间体暂时升高的浓度的阻碍；这具有矛盾的效果，即尽管整体趋势是解离，但有利于那些中间体的重新组装。滞后现象掩盖了除接触能最显著降低之外的所有情况。相比之下，组装反应迅速接近平衡。这些结果首次对病毒衣壳如何在极端条件下保持完整但仍能“呼吸”提供了严格的解释。稳定性增强的生物学意义在于可能需要一个触发事件来启动病毒脱壳。
