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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Phospholamban structural dynamics in lipid bilayers probed by a spin label rigidly coupled to the peptide backbone
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Phospholamban structural dynamics in lipid bilayers probed by a spin label rigidly coupled to the peptide backbone

机译：刚性结合到肽骨架上的自旋标签探测脂质双层中的磷脂酰肌醇的结构动力学

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摘要

We have used chemical synthesis and electron paramagnetic resonance to probe the structural dynamics of phospholamban (PLB) in lipid bilayers. Derivatives of monomeric PLB were synthesized, each of which contained a single spin-labeled 2,2,6,6,-Tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid amino acid, with the nitroxide-containing ring covalently and rigidly attached to the alpha-carbon, providing direct insight into the conformational dynamics of the peptide backbone. 2,2,6,6,-tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid was attached at positions 0, 11, and 24 in the cytoplasmic domain or at position 46 in the transmembrane domain. The electron paramagnetic resonance spectrum of the transmembrane domain site (position 46) indicates a single spectral component corresponding to strong immobilization of the probe, consistent with the presence of a stable and highly ordered transmembrane helix. In contrast, each of the three cytoplasmic domain probes has two clearly resolved spectral components (conformational states), one of which indicates nearly isotropic nanosecond dynamic disorder. For the probe at position 11, an N-terminal lipid anchor shifts the equilibrium toward the restricted component, whereas Mg2+ shifts it in the opposite direction. Relaxation enhancement, due to Ni2+ ions chelated to lipid head-groups, provides further information about the membrane topology of PLB, allowing us to confirm and refine a structural model based on previous NMR data. We conclude that the cytoplasmic domain of PLB is in a dynamic equilibrium between an ordered conformation, which is in direct contact with the membrane surface, and a dynamically disordered form, which is detached from the membrane and poised to interact with its regulatory target.
机译：我们已经使用化学合成和电子顺磁共振来探测脂质双层中的磷脂酰肌醇（PLB）的结构动力学。合成了单体PLB的衍生物，每个衍生物都包含一个自旋标记的2,2,6,6，-四甲基哌啶-N-氧基-1-氨基-4-羧酸氨基酸，含氮氧化物的环共价和刚性连接到α-碳上，可直接了解肽主链的构象动力学。 2,2,6,6，-四甲基-哌啶-N-氧基1-4氨基-4-羧酸连接在胞质结构域的0、11和24位或跨膜结构域的46位。跨膜结构域位点（位置46）的电子顺磁共振谱图表明单个光谱成分对应于探针的强固定化，与存在稳定且高度有序的跨膜螺旋相一致。相反，三个胞质结构域探针中的每一个都具有两个清晰分辨的光谱成分（构象状态），其中之一表明几乎各向同性的纳秒动态紊乱。对于位置11处的探针，N末端脂质锚将平衡移向受限制的组分，而Mg2 +将其向相反的方向移。由于Ni2 +离子被螯合到脂质头基上，弛豫增强提供了有关PLB膜拓扑的更多信息，从而使我们能够基于以前的NMR数据确认和完善结构模型。我们得出结论，PLB的胞质域处于有序构象与膜表面直接接触的动态构象与从膜上脱离并准备与其调节靶点相互作用的动态无序形式之间的动态平衡。

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