Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S21 pinholin protein
Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S21 pinholin protein
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DOI：
10.1016/j.ab.2018.12.003
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发表时间：
2019-02-15
影响因子：
2.9
通讯作者：
Lorigan, Gary A.
中科院分区：
文献类型：
--
作者：
Drew, Daniel L., Jr.;Ahammad, Tanbir;Lorigan, Gary A.
双链DNA噬菌体裂解途径的机制涉及一类形成小孔的膜蛋白——穴蛋白。本研究重点关注一类尚未充分表征的穴蛋白，即 pinholin，噬菌体 phi 21 的 S-21 蛋白是其原型。在这里，我们报告了 S-21 pinholin 野生型 S(21)68 和负显性突变型 (SIRS)-I-21 的首次体外合成，两者均使用固相肽合成制备并使用生物物理技术进行研究。两种形式的 pinholin 都用硝基氧自旋标记进行标记，并成功掺入二细胞和多层囊泡（膜模拟系统）中。圆二色性显示这两种形式均> 80% α螺旋，与基于文献的预测一致。两种形式的pinholin的摩尔椭圆率比θ(222)/θ(208)为1.4，表明双层中的卷曲螺旋三级结构与空穴形成机制模型中提出的低聚步骤一致。 pinholin 的 P-31 固态 NMR 光谱数据表明两种形式的 pinholin 与膜头基有强烈的相互作用。 P-31 NMR 数据具有轴对称线形状，与层状蛋白脂质体脂质模拟物一致。
The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins, the holins. This study focuses on a poorly characterized class of holins, the pinholin, of which the S-21 protein of phage phi 21 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S-21 pinholin, S(21)68, and negative-dominant mutant form, (SIRS)-I-21, both prepared using solid phase peptide synthesis and studied using biophysical techniques. Both forms of the pinholin were labeled with a nitroxide spin label and successfully incorporated into both bicelles and multilamellar vesicles which are membrane mimetic systems. Circular dichroism revealed the two forms were both > 80% alpha helical, in agreement with the predictions based on the literature. The molar ellipticity ratio [theta](222)/[theta](208) for both forms of the pinholin was 1.4, suggesting a coiled-coil tertiary structure in the bilayer consistent with the proposed oligomerization step in models for the mechanism of hole formation. P-31 solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The P-31 NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics.