Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA; Natural Science Division, Campbellsville University, Campbellsville, KY 42718, USA.
Biochim Biophys Acta Biomembr. 2023 Feb;1865(2):184083. doi: 10.1016/j.bbamem.2022.184083. Epub 2022 Nov 9.
The S protein from bacteriophage lambda is a three-helix transmembrane protein produced by the prophage which accumulates in the host membrane during late gene expression. It is responsible for the first step in lysing the host cell at the end of the viral life cycle by multimerizing together to form large pores which permeabilize the host membrane to allow the escape of virions. Several previous studies have established a model for the assembly of holin into functional holes and the manner in which they pack together, but it is still not fully understood how the very rapid transition from monomer or dimer to multimeric pore occurs with such precise timing once the requisite threshold is reached. Here, site-directed spin labeling with a nitroxide label at introduced cysteine residues is used to corroborate existing topological data from a crosslinking study of the multimerized holin by EPR spectroscopy. CW-EPR spectral lineshape analysis and power saturation data are consistent with a three-helix topology with an unstructured C-terminal domain, as well as at least one interface on transmembrane domain 1 which is exposed to the lumen of the hole, and a highly constrained steric environment suggestive of a tight helical packing interface at transmembrane domain 2.
噬菌体 λ 的 S 蛋白是一种由前噬菌体产生的三螺旋跨膜蛋白，在前噬菌体晚期基因表达过程中积累在宿主膜中。它负责在病毒生命周期结束时通过多聚化形成大孔来裂解宿主细胞的第一步，这些孔使宿主膜通透性增加，允许病毒粒子逃逸。以前的几项研究已经建立了 holin 组装成功能孔的模型，以及它们组装在一起的方式，但仍不完全清楚一旦达到必要的阈值，单体或二聚体如何快速转变为多聚体孔，并且具有如此精确的定时。在这里，通过电子顺磁共振波谱法对引入半胱氨酸残基的氮氧自由基标记进行定点自旋标记，以确证通过交联研究多聚化 holin 的现有拓扑数据。CW-EPR 谱线形状分析和功率饱和数据与三螺旋拓扑结构一致，具有无结构的 C 末端结构域，以及至少一个跨膜结构域 1 的界面暴露于孔的腔中，以及高度约束的空间环境表明跨膜结构域 2 处存在紧密的螺旋包装界面。
