Advanced Isotope Aided NMR For CB2 Structural Study

用于 CB2 结构研究的先进同位素辅助 NMR

• 批准号: 6711687
• 负责人: Xiang-Qun Xie
• 金额: $ 29.23万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6711687
• 关键词: Escherichia coli; G protein; affinity chromatography; biological signal transduction; cannabinoid receptor; circular dichroism; computer simulation; high performance liquid chromatography; molecular cloning; nuclear magnetic resonance spectroscopy; physical model; polymerase chain reaction; protein purification; protein structure; radionuclides; receptor binding; receptor coupling; receptor expression; recombinant proteins; structural biology

DESCRIPTION (provided by applicant): Since the discovery of cannabinoid (CB) receptors, cannabinoid research has witnessed rapid and important developments and renaissance. CB2 derived from human promyelicytic leukemia cell is a subtype of cannabinoid receptors. Unlike its closely related sub-type receptor CB1, which is believed to be responsible for the modulation of Q-type Ca2+ and inwardly rectifying K+ channels in CNS, CB2 receptor is expressed in high quantifies in human spleen and tonsils, and is likely to be involved in the signal transduction processes in immune system, Therefore, CB2 receptor can potentially be a target for immuno-treatments. Thus, knowledge of the 3D structure of CB receptors and the further understanding of ligand-receptor interaction will greatly aid in the rational design of specific CB2 ligands possessing potent therapeutic activities, but devoid from the undesirable side effects. However, its intrinsic membrane protein property makes it difficult to crystallize for x-ray study. Direct NMR study is also restricted due to the large protein size and slow correlation time, whereas NMR study of synthetic polypeptide is limited by available length of peptides, and low signal-to-noise (S/N) of natural abundant peptides. The objective of this proposal is to obtain the purified recombinant CB2 protein segments (transmembrane domains, or helix bundles) that are expressed in Escherichia coli (E. coli) (isotope-enriched media) for structural biology determination by NMR and computer modeling. Such studies will provide valuable experimental data to refine a 3D construct of CB2 receptor. In addition, the proposed studies will determine the structural and conformational information of the CB2 receptor segments that will shed light towards the understanding of receptor binding-activating-signaling mechanisms. Eventually, an experimental-based 3D CB2 structure will be more reliable for rational drug design. Overall, the method proposed here represents a novel combined approach of protein engineering, modem isotope aided NMR, and computer modeling for study of G-protein coupled transmembrane receptors (GPCRs) that is a large family of drug targets (approximately 45 percent of the market drugs). The work accomplished through this proposed research can potentially make a significant contribution to cannabinoid research and NMR structural biology, as well as GPCRs in general.

描述(由申请人提供)： 自从发现大麻素(CB)受体以来，大麻素研究经历了快速和重要的发展和复兴。CB2来源于人早幼粒细胞白血病细胞，是大麻素受体的一个亚型。CB2受体与其亲缘关系密切的亚型受体CB1不同，CB2受体在人的脾和扁桃体中高表达，并可能参与免疫系统的信号转导过程，因此CB2受体有可能成为免疫治疗的靶点。因此，了解CB受体的三维结构和进一步了解配体-受体相互作用将有助于合理设计具有有效治疗活性且无不良副作用的特定CB2配体。 然而，其固有的膜蛋白性质给X射线研究的结晶带来了困难。直接核磁共振研究也受到蛋白质大小和关联时间慢的限制，而合成多肽的核磁共振研究受到多肽有效长度和天然多肽的低信噪比(S/N)的限制。该方案的目的是获得纯化的重组CB2蛋白片段(跨膜区或螺旋束)，并在大肠杆菌(E.Coli)(同位素富集液)中表达，用于核磁共振和计算机模拟的结构生物学测定。这些研究将为完善CB2受体的3D结构提供有价值的实验数据。此外，拟议的研究将确定CB2受体片段的结构和构象信息，这将有助于理解受体结合-激活-信号机制。最终，基于实验的3D CB2结构将为合理的药物设计提供更可靠的依据。总之，本文提出的方法代表了蛋白质工程、现代同位素辅助核磁共振和计算机模拟的一种新的组合方法，用于研究G蛋白偶联跨膜受体(GPCRs)这一大家族的药物靶标(约占上市药物的45%)。通过这项拟议研究完成的工作可能会对大麻素研究和核磁共振结构生物学以及一般的GPCRs做出重大贡献。