PDF(Pubmed)
Abstract：
G-protein coupled receptors (GPCRs) are the largest class of membrane proteins encoded in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminal tail (Ctail). Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the Ctail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and Ctail (H8-Ctail) to investigate its structural stability, conformational dynamics, and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix - even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift-derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement NMR. Taken together, our studies reveal the H8-Ctail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.
摘要：
G蛋白偶联受体（GPCRs）是人类基因组中编码的最大一类膜蛋白，具有高度的药物相关性和对人类健康的影响。这些受体共享七个跨膜螺旋的普遍结构，然后是细胞内，两亲性螺旋8（H8）和无序的C末端尾巴（尾巴）。在过去的二十年里，技术进步已经导致了1000多个受体结构，然而，H8和尾巴经常是构象异质的或完全不存在。在这里，我们合成了包含神经降压素受体1（NTS1）H8和尾（H8-尾）的肽，以研究其结构稳定性，构象动力学，以及在洗涤剂和磷脂胶束存在下的取向，模仿膜。圆二色性（CD）和核磁共振（NMR）测量证实，两性离子1,2-二庚酰基-sn-甘油-3-磷酸胆碱是H8结构的有效稳定剂，而常用的支链洗涤剂月桂基麦芽糖新戊二醇（LMNG）无法完全稳定螺旋-即使数量比其临界胶束浓度大四个数量级。然后我们使用NMR光谱来分配主链化学位移。使用一系列温度和脂质滴定将H8边界定义为来自化学位移扰动的F376-R392，共振强度的变化，和化学位移衍生的phi/psi角度。最后,H8方位角和倾斜角，使用顺磁弛豫增强NMR测量定义膜正常的相对螺旋取向。一起来看,我们的研究表明，H8-ctail区域对膜的理化性质敏感，并且比以前通过静态结构技术提出的更具适应性的行为。
