The bacteriophage infection cycle plays a crucial role in recycling the world’s biomass. Bacteriophages devise various cell lysis systems to strictly control the length of the infection cycle for an efficient phage life cycle. Phages evolved with lysis protein systems, which can control and fine-tune the length of this infection cycle depending on the host and growing environment. Among these lysis proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time and concentration hence known as the simplest molecular clock. Pinholin S²¹ is the holin from phage Φ21, which defines the cell lysis time through a predefined ratio of active pinholin and antipinholin (inactive form of pinholin). Active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously we reported the structural dynamics and topology of active pinholin S²¹68. Currently, there is no detailed structural study of the antipinholin using biophysical techniques. In this study, the structural dynamics and topology of antipinholin S²¹68_IRS in DMPC proteoliposomes is investigated using electron paramagnetic resonance (EPR) spectroscopic techniques. Continuous-wave (CW) EPR line shape analysis experiments of 35 different R1 side chains of S²¹68_IRS indicated restricted mobility of the transmembrane domains (TMDs), which were predicted to be inside the lipid bilayer when compared to the N- and C-termini R1 side chains. In addition, the R1 accessibility test performed on 24 residues using the CW-EPR power saturation experiment indicated that TMD1 and TMD2 of S²¹68_IRS were incorporated into the lipid bilayer where N- and C-termini were located outside of the lipid bilayer. Based on this study, a tentative model of S²¹68_IRS is proposed where both TMDs remain incorporated into the lipid bilayer and N- and C-termini are located outside of the lipid bilayer. This work will pave the way for the further studies of other holins using biophysical techniques and will give structural insights into these biological clocks in molecular detail.

中文翻译：

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使用连续波电子顺磁共振波谱研究脂质双层中非活性形式的S21 Holin的结构动力学和拓扑结构。

噬菌体的感染周期在循环利用世界生物质方面起着至关重要的作用。噬菌体设计了各种细胞裂解系统来严格控制感染周期的长度，以实现有效的噬菌体生命周期。噬菌体随着裂解蛋白系统的进化而发展，裂解蛋白系统可以根据宿主和生长环境控制和微调该感染周期的长度。在这些裂解蛋白中，holin通过在等位基因特异性的时间和浓度下透化内膜来控制宿主细胞裂解的第一步和限速步骤，因此被称为最简单的分子钟。品尼高S ²¹是来自噬菌体Φ21的孔蛋白，它通过活性品Pinholin和抗品Pinholin（品醇的非活性形式）的预定比例定义细胞裂解时间。活性品Pinholin和antipinholin通过结构动力学和构象变化来微调裂解时间。先前我们报道了活性品Pinholin S ²¹ 68的结构动力学和拓扑结构。目前，尚无使用生物物理技术对抗品Pinholin进行详细的结构研究。在这项研究中，使用电子顺磁共振（EPR）光谱技术研究了DMPC蛋白脂质体中抗品红蛋白S ²¹ 68 _IRS的结构动力学和拓扑。S ²¹ 68的35个不同R1侧链的连续波（EPW）EPR线形分析实验_IRS指出跨膜域（TMD）的活动受限，与N和C末端R1侧链相比，它被预测在脂质双层内部。此外，使用CW-EPR功率饱和实验对24个残基进行的R1可及性测试表明，S ²¹ 68 _IRS的TMD1和TMD2已掺入N-和C-末端位于脂质双层外部的脂质双层中。根据这项研究，建立了S ²¹ 68 _IRS的暂定模型提出了其中两个TMD均保持结合到脂质双层中并且N末端和C末端位于脂质双层外部的提议。这项工作将为使用生物物理技术进一步研究其他孔蛋白铺平道路，并将在分子细节上对这些生物钟提供结构上的见识。