Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
Nat Protoc. 2019 Aug;14(8):2483-2520. doi: 10.1038/s41596-019-0188-9. Epub 2019 Jul 3.
The transmembrane (TM) anchors of cell surface proteins have been one of the 'blind spots' in structural biology because they are generally very hydrophobic, sometimes dynamic, and thus difficult targets for structural characterization. A plethora of examples show these membrane anchors are not merely anchors but can multimerize specifically to activate signaling receptors on the cell surface or to stabilize envelope proteins in viruses. Through a series of studies of the TM domains (TMDs) of immune receptors and viral membrane proteins, we have established a robust protocol for determining atomic-resolution structures of TM oligomers by NMR in bicelles that closely mimic a lipid bilayer. Our protocol overcomes hurdles typically encountered by structural biology techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM) when studying small TMDs. Here, we provide the details of the protocol, covering five major technical aspects: (i) a general method for producing isotopically labeled TM or membrane-proximal (MP) protein fragments that involves expression of the protein (which is fused to TrpLE) into inclusion bodies and releasing the target protein by cyanogen bromide (CNBr) cleavage; (ii) determination of the oligomeric state of TMDs in bicelles; (iii) detection of intermolecular contacts using nuclear Overhauser effect (NOE) experiments; (iv) structure determination; and (v) paramagnetic probe titration (PPT) to characterize the membrane partition of the TM oligomers. This protocol is broadly applicable for filling structural gaps of many type I/II membrane proteins. The procedures may take 3-6 months to complete, depending on the complexity and stability of the protein sample.
细胞膜蛋白的跨膜（TM）结构域一直是结构生物学的“盲点”之一，因为它们通常具有很强的疏水性，有时还具有动态性，因此难以进行结构特征分析。大量实例表明，这些膜锚不仅是锚定物，而且可以特异性地多聚化，激活细胞表面的信号受体，或稳定病毒包膜蛋白。通过对免疫受体和病毒膜蛋白的 TM 结构域（TMD）进行一系列研究，我们建立了一种在双分子层脂质体中通过 NMR 确定 TM 寡聚体原子分辨率结构的稳健方案。该方案克服了结构生物学技术（如 X 射线晶体学和冷冻电镜（cryo-EM））在研究小 TMD 时遇到的障碍。在这里，我们提供了该方案的详细信息，涵盖了五个主要技术方面：（i）一种用于产生同位素标记 TM 或膜近端（MP）蛋白片段的通用方法，该方法涉及将蛋白（与 TrpLE 融合）表达到包涵体中，并通过溴化氰（CNBr）切割释放目标蛋白；（ii）在双分子层脂质体中确定 TMD 寡聚状态；（iii）使用核奥佛豪瑟效应（NOE）实验检测分子间接触；（iv）结构确定；以及（v）顺磁探针滴定（PPT），以表征 TM 寡聚体的膜分配。该方案广泛适用于填补许多 I/II 型跨膜蛋白的结构空白。这些程序可能需要 3-6 个月才能完成，具体取决于蛋白样品的复杂性和稳定性。
