利用神经紧张素受体 1 肽揭示 G 蛋白偶联受体螺旋 8 的溶液结构和取向。

IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Protein Science Pub Date : 2024-06-01 DOI:10.1002/pro.4976
James B Bower, Scott A Robson, Joshua J Ziarek
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引用次数: 0

摘要

G 蛋白偶联受体(GPCR)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,对人类健康具有重要影响。这些受体的普遍结构是七个跨膜螺旋,然后是细胞内的两性螺旋 8 (H8) 和无序的 C 端尾 (Ctail)。随着技术的进步,过去二十年中已发现了 1000 多种受体结构,但 H8 和 Ctail 在构象上往往不一致或完全不存在。在这里,我们合成了一种由神经紧张素受体 1(NTS1)H8 和 Ctail(H8-Ctail)组成的多肽,以研究其在洗涤剂和磷脂胶束(模拟膜)存在下的结构稳定性、构象动力学和定向。圆二色性(CD)和核磁共振(NMR)测量证实,齐聚物 1,2-二庚酰-sn-甘油-3-磷酸胆碱是 H8 结构的强效稳定剂,而常用的支链洗涤剂十二烷基麦芽糖新戊二醇(LMNG)则无法完全稳定螺旋,即使其用量比临界胶束浓度高出四个数量级。我们随后使用核磁共振光谱来确定骨架化学位移。通过一系列温度和脂质滴定,我们利用化学位移扰动、共振强度变化和化学位移衍生的 phi/psi 角将 H8 边界定义为 F376-R392。最后,利用顺磁弛豫增强核磁共振测量了 H8 的方位角和倾斜角,确定了螺旋相对于膜法线的方向。总之,我们的研究揭示了 H8-Ctail 区域对膜理化特性的敏感性,它比以前的静态结构技术所显示的具有更强的适应性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Insights on the G protein-coupled receptor helix 8 solution structure and orientation using a neurotensin receptor 1 peptide.

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.

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来源期刊
Protein Science
Protein Science 生物-生化与分子生物学
CiteScore
12.40
自引率
1.20%
发文量
246
审稿时长
1 months
期刊介绍: Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution. Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics. The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication. Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).
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