Enrichment of Membrane Proteins for Downstream Analysis Using Styrene Maleic Acid Lipid Particles (SMALPs) Extraction.
Enrichment of Membrane Proteins for Downstream Analysis Using Styrene Maleic Acid Lipid Particles (SMALPs) Extraction.
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DOI：
10.21769/bioprotoc.4728
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发表时间：
2023-08-05
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影响因子：
0.8
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整合膜蛋白是一类重要的细胞蛋白。这些参与关键的细胞过程，如信号转导受体转运，许多在质膜内运作。超过一半的FDA批准的蛋白质靶向药物通过与含有至少一个跨膜区域的蛋白质相互作用来发挥作用，但其与治疗药物的天然相互作用的表征和研究仍然是一个重大挑战。这一挑战部分是由于这些蛋白质通常以少量存在于细胞内。膜蛋白的有效溶解也是一个问题，通常用于溶解膜的去污剂会导致功能活性和关键相互作用伙伴的丧失。近年来，已经研究了在其天然脂质环境中提取膜蛋白的替代方法，目的是产生功能性纳米盘，维持蛋白质-蛋白质和蛋白质-脂质相互作用。一个有前途的方法涉及提取膜蛋白的苯乙烯马来酸脂质颗粒（SMALPs）的形式，允许保留其天然构象。这种提取方法为进一步的蛋白质分析提供了许多优势，并允许研究蛋白质与其他分子（如药物）的相互作用。在这里，我们描述了一种有效的SMALP提取功能活性膜蛋白复合物内纳米盘的协议。我们展示的方法上分离的低拷贝数质膜受体复合物，烟碱乙酰胆碱受体（nAChR），从成年果蝇头。我们证明，这些nanodisks可用于研究天然受体-配体相互作用。该方案可以应用于许多生物学场景，以提取低拷贝数整合膜蛋白的天然构象。
Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein–protein and protein–lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor–ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.