Ross P D, Black L W, Bisher M E, Steven A C
J Mol Biol. 1985 Jun 5;183(3):353-64. doi: 10.1016/0022-2836(85)90006-3.
Inter- and intra-subunit bonding within the surface lattice of the capsid of bacteriophage T4 has been investigated by differential scanning calorimetry of polyheads, in conjunction with electron microscopy, limited proteolysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The bonding changes corresponding to successive stages of assembly of the major capsid protein gp23, including its maturation cleavage, were similarly characterized. The uncleaved/unexpanded surface lattice exhibits two endothermic transitions. The minor event, at 46 degrees C, does not visibly affect the surface lattice morphology and probably represents denaturation of the N-terminal domain of gp23. The major endotherm, at 65 degrees C, represents denaturation of the gp23 polymers. Soluble gp23 from dissociated polyheads is extremely unstable and exhibits no endotherm. Cleavage of gp23 to gp23* and the ensuing expansion transformation effects a major stabilization of the surface lattice of polyheads, with single endotherms whose melting temperatures (tm) range from 73 to 81 degrees C, depending upon the mutant used and the fraction of gp23 that is cleaved to gp23 prior to expansion. Binding of the accessory proteins soc and hoc further modulates the thermograms of cleaved/expanded polyheads, and their effects are additive. hoc binding confers a new minor endotherm at 68 degrees C corresponding to at least partial denaturation of hoc. Denatured hoc nevertheless remains associated with the surface lattice, although in an altered, protease-sensitive state which correlates with delocalization of hoc subunits visualized in filtered images. While hoc binding has little effect on the thermal stability of the gp23* matrix, soc binding further stabilizes the surface lattice (delta Hd approximately +50%; delta tm = +5.5 degrees C). It is remarkable that in all states of the surface lattice, the inter- and intra-subunit bonding configurations of gp23 appear to be co-ordinated to be of similar thermal stability. Thermodynamically, the expansion transformation is characterized by delta H much less than 0; delta Cp approximately 0, suggesting enhancement of van der Waals' and/or H-bonding interactions, together with an increased exposure to solvent of hydrophobic residues of gp23 in the expanded state. These findings illuminate hypotheses of capsid assembly based on conformational properties of gp23: inter alia, they indicate a role for the N-terminal portion of gp23 in regulating polymerization, and force a reappraisal of models of capsid swelling based on the swivelling of conserved domains.
通过对多角体进行差示扫描量热法，并结合电子显微镜、有限蛋白酶解和十二烷基硫酸钠/聚丙烯酰胺凝胶电泳，研究了噬菌体T4衣壳表面晶格内亚基间和亚基内的结合。对主要衣壳蛋白gp23组装的连续阶段（包括其成熟切割）所对应的结合变化进行了类似的表征。未切割/未扩展的表面晶格呈现出两个吸热转变。较小的转变发生在46℃，对表面晶格形态没有明显影响，可能代表gp23 N端结构域的变性。主要的吸热峰出现在65℃，代表gp23聚合物的变性。从解离的多角体中获得的可溶性gp23极其不稳定，且无吸热峰。将gp23切割为gp23以及随后的扩展转变使多角体的表面晶格得到显著稳定，呈现出单一的吸热峰，其解链温度（tm）范围为73至81℃，这取决于所使用的突变体以及在扩展前被切割为gp23的gp23的比例。辅助蛋白soc和hoc的结合进一步调节了切割/扩展多角体的热谱图，且它们的作用是相加的。hoc的结合在68℃赋予一个新的较小吸热峰，对应于hoc至少部分变性。尽管变性的hoc仍与表面晶格相关联，但其处于一种改变的、对蛋白酶敏感的状态，这与在过滤图像中观察到的hoc亚基的离域化相关。虽然hoc的结合对gp23基质的热稳定性影响不大，但soc的结合进一步稳定了表面晶格（ΔHd约增加50%；Δtm = +5.5℃）。值得注意的是，在表面晶格的所有状态下，gp23的亚基间和亚基内结合构型似乎协同作用，具有相似的热稳定性。从热力学角度来看，扩展转变的特征是ΔH远小于0；ΔCp约为0，这表明范德华力和/或氢键相互作用增强，同时在扩展状态下gp23的疏水残基与溶剂的接触增加。这些发现阐明了基于gp23构象特性的衣壳组装假说：特别是，它们表明gp23的N端部分在调节聚合中起作用，并促使人们重新评估基于保守结构域旋转的衣壳膨胀模型。
