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

根据分子模拟研究，这些配体(SWR-0342SA)与β受体(β-AR)的结合部位是β_1受体中的Asp104和Leu335，以及β_3受体中的Asp117、Ser209、Leu303和Ser192。用放射配基结合分析方法测定了这些化合物的亲和力。此外，我们还用核磁共振技术分析了这些化合物与β-AR的相互作用部位。β_1-肾上腺素能受体的3^&lt；rd&gt；tmd序列为KKKELWTSVDVLAVTASIETLAVIALDRYLAKK。该多肽由Sigma Genosys合成并购买，经高效液相色谱鉴定纯度达74%。将纯化后的多肽溶解于DMSO溶液中，制成核磁共振样品溶液。在JEOL 500 MHz核磁共振机上进行了一维核磁共振和二维核磁共振实验。一维核磁共振实验采用预饱和法，二维核磁共振实验包括双量子滤波相关谱(DQFCOSY)、核Overhaser效应(NOESY)、旋转标架Overhauser谱(ROESY)、异核多量子相干(HMQC)和异核多键关联(HMBC)。~1H核磁共振波谱的xOffset和xSweep分别为5.8ppm和12ppm。在x_point为95ppm，x_Sweep为200ppm的条件下，还进行了~(13)C核磁共振波谱分析。在NOESY实验中，混合时间为1.872秒。分别获得了β_1-AR的波旁吲哚和33个氨基酸的3^&lt；Rd&Gt；TMD的核磁共振谱。最后，我们对博吲哚与β_1-AR的3^&lt；Rd&Gt；TMD多肽的络合物进行了核磁共振分析。通过与波旁吲哚的结合和非结合的核磁共振波谱，可以得到33^&lt；Rd&gt；TMD多肽的构象结构。

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.