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

经分子模拟研究，这些配体（SWR-0342 SA）与β-肾上腺素受体（β-AR）的结合位点分别为β_1受体的Asp 104和Leu 335，β_3受体的Asp 117、Ser 209、Leu 303和Ser 192。还通过放射性配体结合测定法测定了这些化合物的亲和力。此外，我们还通过核磁共振分析了这些化合物与β-AR的相互作用位点。β<rd>_1-肾上腺素能受体3^ TMD肽序列为KKKELWTSVDVLAVTASIETLAVIALDRYLAK。合成肽并从Sigma Genosys购买，并纯化至通过HPLC判断的&gt;74%纯度。通过将纯化的肽溶解在DMSO溶液中来制备用于NMR样品溶液的纯化的肽。在JEOL 500 MHz核磁共振仪上进行了1D-HNMR和2D-NMR实验。1DNMR实验采用预饱和法，2DNMR实验采用双量子滤波相关谱（DQFCOSY）、核Overhauser效应（NOESY）、旋转标架Overhauser谱（ROESY）、杂原子多量子相干（HMQC）和杂原子多键相关（HMBC）。在x_offset和x_sweep分别为5.8 ppm和12 ppm的情况下进行1H NMR光谱。还<13>进行13 C NMR光谱，其中x_point为95 ppm且x_sweep为200 ppm。在NOESY实验中，混合时间为1.872秒。分别获得了博吲哚洛尔和β_1-AR的3^ TMD的33个氨基酸肽段的NMR谱<rd>。最后，我们对博吲哚洛尔与β_1-AR的3^ TMD肽段的复合物进行了核磁共振分析<rd>。我们可以<rd>从结合和未结合的核磁共振谱中得到33^ 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.