Analysis of 3-dimentional structure β-adrenoceptor ligands and receptor complex by NMR

基于分子模拟研究，这些配体（SWR-0342SA）与β-肾上腺素受体（β-AR）的结合位点分别是β_1受体中的Asp104和Leu335，以及β_3-肾上腺素受体中的Asp117、Ser209、Leu303和Ser192。这些化合物的亲和力也通过放射性配体结合测定法确定。此外，我们还通过NMR分析了这些化合物与β-AR的相互作用位点。β_1-肾上腺素能受体3^<rd> TMD肽序列为KKKELWTSVDVLAVTASIETLAVIALDRYLAKK。该肽由Sigma gensys公司合成并购买，经高效液相色谱法纯化，纯度为bb0 - 74%。将纯化的多肽溶解于DMSO溶液中制备NMR样品溶液。在JEOL 500MHz核磁共振机上进行一维核磁共振和二维核磁共振实验。在一维HNMR实验中采用了预饱和法，而2DNMR实验包括DQFCOSY（双量子滤波相关光谱）、NOESY（核Overhauser效应）、ROESY（旋转框架Overhauser光谱）、HMQC（七核多量子相干）和HMBC（七核多键相关）。在x_offset和x_sweep分别为5.8 ppm和12 ppm的情况下进行了^1H NMR波谱分析。在x_point为95 ppm， x_sweep为200 ppm的情况下，也进行了^<13>C核磁共振波谱。在noesi实验中，混合时间为1.872秒，分别获得了bindindoll和β_1-AR的3^<rd> TMD的33个氨基酸肽的核磁共振波谱。最后，我们想对bopinddolol与β_1-AR的3^<rd> TMD肽之间的配合物进行NMR分析。通过与bopinddolol的结合和非结合核磁共振波谱，我们可以得到33^<rd> TMD肽的构象结构。

Based on the molecular modeling studies, the binding sites between these ligands (SWR-0342SA) and β-adrenoceptors (β-AR) were Asp104 and Leu335 in the β_1-receptors, and Asp117,Ser209,Leu303 and Ser192 and in β_3-adrenoceptors. Potencies of affinities of these compounds were also determined by the radioligand binding assay method. In addition, we analyzed the interaction sites between these compounds and β-AR by NMR. The sequence of 3^<rd> TMD peptide of the β_1- adrenergic receptor was found KKKELWTSVDVLAVTASIETLAVIALDRYLAKK. The peptide was synthesized and purchased from Sigma Genosys and purified to >74% purity as judged by HPLC. The purified peptide was prepared for NMR sample solution by dissolving it in DMSO solution. 1D HNMR and 2D NMR experiment were done in the JEOL 500MHz NMR machine. In 1D HNMR experiment presaturation method was done, whereas 2DNMR experiment was included DQFCOSY (double quantum filtered correlation spectroscopy), NOESY (Neclear Overhauser effects), ROESY (Rotating frame Overhauser spectroscopy), HMQC (hetaronuclear Multiple Quantum coherence) and HMBC (Hetaronuclear Multiple Bond Correlation). The ^1H NMR spectroscopy was performed where x_offset and x_sweep was 5.8 ppm and 12 ppm respectively. The ^<13>C NMR spectroscopy was also done where x_point was 95 ppm and x_sweep was 200 ppm. In NOESY experiment mixing time was 1.872 sec. The NMR spectra of bopindolol and 33 amino acid peptide of 3^<rd> TMD of β_1-AR were obtained individually. Finally we want to perform NMR analysis of complex between bopindolol and 3^<rd> TMD peptide of β_1-AR. We may be able to get the conformational structure of 33^<rd> TMD peptide from the bound and unbound NMR spectroscopy with bopindolol.