STRUCTURAL TRANSITIONS DURING BACTERIOPHAGE-HK97 HEAD ASSEMBLY
STRUCTURAL TRANSITIONS DURING BACTERIOPHAGE-HK97 HEAD ASSEMBLY
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DOI：
10.1006/jmbi.1995.0168
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发表时间：
1995-04-07
影响因子：
5.6
通讯作者：
HENDRIX, RW
中科院分区：
文献类型：
--
作者：
DUDA, RL;HEMPEL, J;HENDRIX, RW
噬菌体HK 97从42 kDa的主要头部蛋白质构建其头部壳，但在成熟头部中既没有发现该42 kDa蛋白质也没有发现其加工的31 kDa形式。相反，每个主要的头蛋白亚基通过体内和体外发生的蛋白质交联反应共价交联成五个、六个或更多个的寡聚体。阻断前体成熟的突变体导致两种类型的前体之一的积累，称为前体I和前体II。Prohead I由约415个拷贝的42 kDa（384个氨基酸）蛋白质亚基组装而成，并在突变型amU 4的感染中积累。组装后，每个亚基的N-末端102个氨基酸被去除，留下由31 kDa亚基构成的前头壳，称为前头II，其在突变体amC 2的感染中积累。在DNA包装过程中，当头壳膨胀时，所有的头蛋白亚基都与其他亚基共价交联。纯化的Prohead II（或不完全的Prohead I）在体外响应于诱导壳膨胀的多种条件中的任何一种而交联，包括通常用于蛋白质分析的条件。在体外交联有效地发生在没有添加辅因子的酶，我们建议，交联催化壳亚基本身。通过观察琼脂糖凝胶中Prohead II电泳迁移率的降低，可以很容易地监测外壳膨胀。使用琼脂糖凝胶中的迁移率变化来监测扩展和SDS/凝胶电泳来监测体外交联，我们发现扩展先于交联，并且是交联所需的，并且我们建议扩展触发交联反应。从Prohead II分离的肽和体外交联的Prohead II的比较显示了一个单一的改变的主要交联肽，其中一个赖氨酸，来自蛋白质序列的赖氨酸169，连接到天冬酰胺356，推测来自相邻的亚基。通过质谱法对含有交联的肽的检查表明，交联键是赖氨酸和天冬酰胺残基的侧链之间的酰胺。
Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant amC2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro, we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.