Assembly-associated structural changes of bacteriophage T7 capsids. Detection by use of a protein-specific probe.
Assembly-associated structural changes of bacteriophage T7 capsids. Detection by use of a protein-specific probe.
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噬菌体 T7 衣壳的组装相关结构变化。
DOI：
10.1016/s0006-3495(92)81724-1
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发表时间：
1992
影响因子：
3.4
通讯作者：
Serwer,P
中科院分区：
文献类型：
--
作者：
Khan,SA;Griess,GA;Serwer,P
为了检测当预组装的噬菌体T7衣壳包装和切割成成熟尺寸的更长的（串联）DNA时发生的衣壳结构变化，本文确定了蛋白质特异性探针1,1'-双（4-苯胺）naphthale -5,5'-二磺酸（bisans）与T7噬菌体T7结合的动力学和热力学，T7 DNA缺失（8.4%）突变体。以及一种无DNA的T7衣壳（甲硝唑酰胺低密度衣壳II），已知它是一种DNA包装中间体，具有相关衣壳（甲硝唑酰胺高密度衣壳II）中不存在的渗透性屏障。最初，一些与噬菌体或甲硝唑胺低密度衣壳II的结合发生得太快而无法量化（阶段1，持续时间< 10秒）。随后的结合（阶段2）以一级动力学发生。甲咪唑胺高密度衣壳II只发生1期结合。这些观察结果，以及乙啶对结合的双ans荧光猝灭的动力学和双ans诱导的蛋白质改变的性质，都可以用第二阶段结合发生在内部位点的假设来解释。这些内部位点的数量随着包装DNA密度的降低而增加。伴随而来的结构变化可能是引发串联体解理的信号。还获得了以下证据：(a)以前未检测到的包装相关的外衣壳外壳主要蛋白构象的变化和(b) T7衣壳内部的渗透性屏障的分配。
To detect changes in capsid structure that occur when a preassembled bacteriophage T7 capsid both packages and cleaves to mature-size longer (concatameric) DNA, the kinetics and thermodynamics are determined here for the binding of the protein-specific probe, 1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid (bis-ANS), to bacteriophage T7, a T7 DNA deletion (8.4%) mutant, and a DNA-free T7 capsid (metrizamide low density capsid II) known to be a DNA packaging intermediate that has a permeability barrier not present in a related capsid (metrizamide high density capsid II). Initially, some binding to either bacteriophage or metrizamide low density capsid II occurs too rapidly to quantify (phase 1, duration < 10 s). Subsequent binding (phase 2) occurs with first-order kinetics. Only the phase 1 binding occurs for metrizamide high density capsid II. These observations, together with both the kinetics of the quenching by ethidium of bound bis-ANS fluorescence and the nature of bis-ANS-induced protein alterations, are explained by the hypothesis that the phase 2 binding occurs at internal sites. The number of these internal sites increases as the density of the packaged DNA decreases. The accompanying change in structure is potentially the signal for initiating cleavage of a concatemer. Evidence for the following was also obtained: (a) a previously undetected packaging-associated change in the conformation of the major protein of the outer capsid shell and (b) partitioning by a permeability barrier of the interior of the T7 capsid.