Characterizing the Locus of a Peripheral Membrane Protein–Lipid Bilayer Interaction Underlying Protein Export Activity in E. coli
Characterizing the Locus of a Peripheral Membrane Protein–Lipid Bilayer Interaction Underlying Protein Export Activity in E. coli
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表征大肠杆菌中蛋白质输出活性的外周膜蛋白基因座 - 脂质双层相互作用
DOI：
10.1021/acs.langmuir.9b03606
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发表时间：
2020
期刊：
影响因子：
3.9
通讯作者：
King, Gavin M.
中科院分区：
文献类型：
--
作者：
Matin, Tina R.;Utjesanovic, Milica;Sigdel, Krishna P.;Smith, Virginia F.;Kosztin, Ioan;King, Gavin M.
外周膜蛋白-脂质双层相互作用强度的定量表征是理解许多蛋白质靶向途径的基础。SecA是一种外周膜蛋白，在前体蛋白跨E.杆菌SecA的N-末端的膜结合活性对于移位酶功能是至关重要的。然而，相互作用的机械强度和动力学途径，这段SecA的经验时，在附近的一个E。共极性脂质双层尚未被表征。我们直接测量的N-末端SecA脂质双层相互作用，使用精密单分子原子力显微镜（AFM）为基础的动态力谱。为了提供AFM无法获得的构象数据，我们还进行了全原子分子动力学模拟和圆二色性测量。SecA的N-末端10个氨基酸在与两性离子脂质头部基团结合时几乎没有二级结构，但是当带负电荷的脂质存在时，出现了使脂质结合的蛋白质片段刚性化的二级结构。单分子蛋白质-脂质解离数据的分析收敛于在不存在力的情况下的明确定义的脂质结合态寿命，τ 0 lipid = 0.9 s，其与分泌过程的基本时间尺度很好地分离并且长于分泌过程的基本时间尺度，所述基本时间尺度被定义为移位单个氨基酸残基所需的时间（约50 ms）。由于包括此处采用的最小系统在内的因素，τ 0 lipidis的该值可能代表体内膜结合寿命的下限。
Quantitative characterization of the strength of peripheral membrane protein–lipid bilayer interactions is fundamental in the understanding of many protein targeting pathways. SecA is a peripheral membrane protein that plays a central role in translocating precursor proteins across the inner membrane ofE. coli. The membrane binding activity of the extreme N-terminus of SecA is critical for translocase function. Yet, the mechanical strength of the interaction and the kinetic pathways that this segment of SecA experiences when in proximity of anE. colipolar lipid bilayer has not been characterized. We directly measured the N-terminal SecA-lipid bilayer interaction using precision single molecule atomic force microscope (AFM)-based dynamic force spectroscopy. To provide conformational data inaccessible to AFM, we also performed all-atom molecular dynamics simulations and circular dichroism measurements. The N-terminal 10 amino acids of SecA have little secondary structure when bound to zwitterionic lipid head groups, but secondary structure, which rigidifies the lipid-bound protein segment, emerges when negatively charged lipids are present. Analysis of the single molecule protein–lipid dissociation data converged to a well-defined lipid-bound-state lifetime in the absence of force, τ0lipid= 0.9 s, which is well separated from and longer than the fundamental time scale of the secretion process, defined as the time required to translocate a single amino acid residue (∼50 ms). This value of τ0lipidis likely to represent a lower limit of thein vivomembrane-bound lifetime due to factors including the minimal system employed here.