Center for Structural Biology, Vlaams Instituut voor Biotechnologie, Brussels, Belgium; Structural Biology Brussels, Department of Bioengineering Sciences, VUB, Brussels, Belgium.
Center for Structural Biology, Vlaams Instituut voor Biotechnologie, Brussels, Belgium; Structural Biology Brussels, Department of Bioengineering Sciences, VUB, Brussels, Belgium.
J Biol Chem. 2024 Oct;300(10):107734. doi: 10.1016/j.jbc.2024.107734. Epub 2024 Sep 2.
Ryanodine receptors (RyRs) are large Ca release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within 4 h on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bovine RyR2 reconstituted in nanodisc, and mouse RyR2 in detergent. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in cases where the amount of starting material is limited.
Ryanodine 受体（RyRs）是位于内质网或肌质网膜中的大型钙释放通道。哺乳动物中已鉴定出三种 RyR 同工型，其功能障碍与一系列危及生命的疾病有关。大量天然组织或真核细胞培养的需求限制了 RyRs 结构研究的进展。在这里，我们报告了一种利用纳米体从仅 5 毫克总蛋白中纯化 RyRs 的方法。该纯化过程从分离的膜到 cryo-EM 级别的蛋白质，在实验台上 4 小时内完成，可获得用于 cryo-EM 分析的蛋白质。这通过解决兔 RyR1 的结构来证明，该蛋白在去污剂中溶解，重构成脂质纳米盘或脂质体，以及牛 RyR2 在纳米盘中重构成鼠 RyR2 在去污剂中。所报道的方法促进了针对药物开发的 RyRs 结构研究，并且在起始材料量有限的情况下非常有用。
