Department of Chemistry and Biochemistry , Miami University , Oxford , Ohio 45056 , United States.
J Phys Chem B. 2019 Sep 26;123(38):8048-8056. doi: 10.1021/acs.jpcb.9b06480. Epub 2019 Sep 16.
Pinholin S68 is an essential part of the phage Φ21 lytic protein system to release the virus progeny at the end of the infection cycle. It is known as the simplest natural timing system for its precise control of hole formation in the inner cytoplasmic membrane. Pinholin S68 is a 68 amino acid integral membrane protein consisting of two transmembrane domains (TMDs) called TMD1 and TMD2. Despite its biological importance, structural and dynamic information of the S68 protein in a membrane environment is not well understood. Systematic site-directed spin labeling and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopic studies of pinholin S68 in 1,2-dimyristoyl--glycero-3-phosphocholine (DMPC) proteoliposomes are used to reveal the structural topology and dynamic properties in a native-like environment. CW-EPR spectral line-shape analysis of the R1 side chain for 39 residue positions of S68 indicates that the TMDs have more restricted mobility when compared to the N- and C-termini. CW-EPR power saturation data indicate that TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S68. A tentative structural topology model of pinholin S68 is also suggested based on EPR spectroscopic data reported in this study.
Pinholin S68 是噬菌体 Φ21 裂解蛋白系统的重要组成部分，可在感染周期结束时释放病毒后代。由于其对内质膜孔形成的精确控制，Pinholin S68 被称为最简单的天然定时系统。Pinholin S68 是一种由 68 个氨基酸组成的完整膜蛋白，由两个跨膜结构域（TMDs）组成，称为 TMD1 和 TMD2。尽管它具有重要的生物学意义，但 Pinholin S68 在膜环境中的结构和动态信息仍未得到很好的理解。在 1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱（DMPC）脂质体中，通过系统的定点自旋标记和连续波电子顺磁共振（CW-EPR）光谱研究，揭示了在天然环境中的结构拓扑和动态特性。Pinholin S68 的 R1 侧链的 CW-EPR 谱线形状分析表明，与 N-和 C-末端相比，TMDs 的流动性受到更多限制。CW-EPR 功率饱和数据表明，TMD1 部分从脂质双层外部化，并与膜表面相互作用，而 TMD2 在 Pinholin S68 的活性构象中仍然埋在脂质双层中。根据本研究中报道的 EPR 光谱数据，还提出了 Pinholin S68 的暂定结构拓扑模型。
