Advanced Isotope Aided NMR For CB2 Structural Study

描述（由申请人提供）： 自大麻素受体发现以来，大麻素的研究得到了迅速而重要的发展和复兴。CB 2是大麻素受体的一种亚型，来源于人早幼粒细胞白血病细胞。CB 2受体在人的脾脏和扁桃体中有高表达，可能参与免疫系统的信号转导过程，因此，CB 2受体有可能成为免疫治疗的靶点。因此，CB受体的三维结构的知识和配体-受体相互作用的进一步理解将大大有助于具有有效的治疗活性，但没有不良副作用的特定CB 2配体的合理设计。 然而，其固有的膜蛋白性质使其难以结晶用于X射线研究。直接NMR研究也受到限制，由于大的蛋白质尺寸和缓慢的相关时间，而合成多肽的NMR研究受到肽的可用长度和天然丰富肽的低信噪比（S/N）的限制。 本研究的目的是获得纯化的重组CB 2蛋白片段（跨膜结构域或螺旋束），并在大肠杆菌（E. coli）中表达。coli）（同位素富集培养基）用于通过NMR和计算机建模进行结构生物学测定。这些研究将为完善CB 2受体的3D构建提供有价值的实验数据。此外，拟议的研究将确定CB 2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-信号传导机制。最终，基于实验的3D CB 2结构将更可靠地用于合理的药物设计。总的来说，这里提出的方法代表了一种新的蛋白质工程，现代同位素辅助NMR和计算机建模的组合方法，用于研究G蛋白偶联跨膜受体（GPCR），这是一个大家族的药物靶点（约45%的市场药物）。通过这项拟议的研究完成的工作可能会对大麻素研究和NMR结构生物学以及一般的GPCR做出重大贡献。

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.