Engineering robust chaperone substrates for refolding studies using the highly bioluminescent protein NanoLuc
Engineering robust chaperone substrates for refolding studies using the highly bioluminescent protein NanoLuc
复制标题
使用高生物发光蛋白 NanoLuc 设计用于重折叠研究的稳健伴侣底物
DOI：
10.1016/j.bpj.2021.11.1138
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发表时间：
2022
影响因子：
3.4
通讯作者：
Marszalek, Piotr E.
中科院分区：
文献类型：
--
作者：
Apostolidou, Dimitra;Marszalek, Piotr E.
真核生物具有通过糖脂锚糖基磷脂酰肌醇（GPIs）锚定到细胞质膜的蛋白质。GPI锚定蛋白（GPI-AP）通过蛋白质-蛋白质相互作用在细胞的保护、调节和活化中发挥作用。GPIs和GPI-AP的研究受到难以分离和产生这些纯形式的复杂分子的阻碍。在本文中，我们应用半合成方法通过结合蛋白质表达和合成GPI与蛋白质的化学选择性连接来合成定义明确的GPI-AP。具体而言，我们将表达伯氏疟原虫裂殖子表面蛋白1（MSP 1 -19）的19 kDa片段，并将其连接到合成的GPI。GPI锚定的MSP 1 -19将被插入到不同组成的细胞大小的模型膜（巨大的单层囊泡）中，并插入到支持的脂质双层中，以使用各种基于荧光显微镜的技术和表面等离子体共振来研究GPI对MSP 1 -19锚定蛋白的作用。MSP 1 -19蛋白富含半胱氨酸。评估其折叠是研究MSP 1 -19如何整合到模型膜中以及GPI锚的结构如何影响这一点的必要前提。我们利用基于NMR的方法来研究蛋白质在表达和连接后的折叠。
Eukaryotes have proteins anchored to the cells’ plasma membrane via the glycolipid anchor glycosylphosphatidylinositol (GPIs). GPI anchored proteins (GPI-APs) can play a role in the protection, regulation and activation of cells via protein-protein interactions. Studies of GPIs and GPI-APs are hindered by the difficulty of isolating and producing these complex molecules in pure forms. Herein, we apply a semi-synthetic method to synthesize well-defined GPI-APs by combining protein expression and chemo-selective attachment of synthetic GPI to proteins. Specifically, we will express the 19 kDa fragment of the Plasmodium berghei merozoite surface protein 1 (MSP1-19) and ligate it to a synthetic GPI. The GPI-anchored MSP1-19 will be inserted into cell-sized model membranes (giant unilamellar vesicles) of different composition and into supported lipid bilayers to investigate the role of GPIs on MSP1-19 anchored protein using various fluorescence microscopy-based techniques and surface plasmon resonance. MSP1-19 protein is rich in cysteine. Evaluating its folding is a warranted prerequisite to studying, how MSP1-19 integrates into model membranes and how the structure of the GPI anchor affects this. We utilize a NMR based approach to study the protein folding post expression and ligation.