Kinetic and calorimetric evidence for two distinct scaffolding protein binding populations within the bacteriophage P22 procapsid.
Kinetic and calorimetric evidence for two distinct scaffolding protein binding populations within the bacteriophage P22 procapsid.
复制标题
噬菌体 P22 衣壳内两个不同支架蛋白结合群体的动力学和量热证据。
DOI：
10.1021/bi0026167
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
PreveligeJr,PE
中科院分区：
文献类型：
--
作者：
Parker,MH;Brouillette,CG;PreveligeJr,PE
各种各样的病毒需要瞬时存在的支架蛋白来指导衣壳组装。在噬菌体P22的情况下，已经提出了一个模型，在该模型中，支架蛋白选择性地稳定在途径上生长的中间体。用光散射和等温滴定量热法分析了P22噬菌体支架蛋白与前衣壳蛋白结合的化学计量和热力学。在10至37℃之间进行的量热实验与至少存在两组不同的结合位点是一致的，这与通过光散射分析获得的动力学证据是一致的。与高亲和力部位的结合发生在20℃，每个前衣壳的化学计量比约为60个支架分子，表观Kd值约为100−300 nm，几乎完全由焓驱动。对于第二结合群体，由于结合热很小，不可能对数据进行精确拟合，但结合的热力学明显不同于高亲和力相。结合热容变化(ΔCP)对高亲和力位点较大，对两组结合热容变化均为负。氯化钠(1M)的加入极大地降低了表观ΔH的大小，这与先前的证据一致，即静电相互作用在结合中起主要作用。一种突变的支架蛋白，形成只与高亲和力位点结合的共价二聚体(R74C/L177I)。这些数据包括对外壳蛋白/支架蛋白相互作用的能量学的第一次定量测量。
A wide variety of viruses require the transient presence of scaffolding proteins to direct capsid assembly. In the case of bacteriophage P22, a model in which the scaffolding protein selectively stabilizes on-pathway growing intermediates has been proposed. The stoichiometry and thermodynamics of binding of the bacteriophage P22 scaffolding protein within the procapsid were analyzed by light scattering and isothermal titration calorimetry. Calorimetric experiments carried out between 10 and 37 °C were consistent with the presence of at least two distinct populations of binding sites, in agreement with kinetic evidence obtained by a light scattering assay. Binding to the high-affinity sites occurred at 20 °C with a stoichiometry of approximately 60 scaffolding molecules per procapsid and an apparentKdof approximately 100−300 nM and was almost completely enthalpy-driven. For the second binding population, precise fitting of the data was impossible due to small heats of binding, but the thermodynamics of binding were clearly distinct from the high-affinity phase. The heat capacity change (ΔCp) of binding was large for the high-affinity sites and negative for both sets of sites. Addition of sodium chloride (1 M) greatly reduced the magnitude of the apparent ΔH, in agreement with previous evidence that electrostatic interactions play a major role in binding. A mutant scaffolding protein that forms covalent dimers (R74C/L177I) bound only to the high-affinity sites. These data comprise the first quantitative measurements of the energetics of the coat protein/scaffolding protein interaction.