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首页> 外文期刊>Biochemistry >Dynamic nuclear polarization study of inhibitor binding to the M2 18-60 proton transporter from influenza A
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Dynamic nuclear polarization study of inhibitor binding to the M2 18-60 proton transporter from influenza A

机译：抑制剂与甲型流感M2 18-60质子转运蛋白结合的动态核极化研究

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摘要

We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M2_(18-60) from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore. Futhermore, the ~(15)N-labeled ammonium of rimantadine was observed near A30 13Cβ and G34 13Cα, suggesting a possible hydrogen bond to A30 carbonyl. Cryogenic DNP was required to observe the weaker external binding site(s) in a ZF-TEDOR spectrum. This approach is generally applicable, particularly for weakly bound ligands, in which case the application of MAS NMR dipolar recoupling requires the low temperatures to quench dynamic exchange processes. For the fully protonated samples investigated, we observed DNP signal enhancements of ～10 at 400 MHz using only 4-6 mM of the polarizing agent TOTAPOL. At 600 MHz and with DNP, we measured a distance between the drug and the protein to a precision of 0.2 ?.
机译：我们展示了使用动态核极化（DNP）阐明配体结合使用偶极耦合魔术角旋转（MAS）NMR的膜蛋白。尤其是，我们使用室温和低温DNP的再偶联实验，检测了甲型流感病毒质子转运蛋白M2_（18-60）中的药物结合。结果表明金刚乙胺的孔结合位点与先前报道的广泛的化学位移变化相关，表明孔中的功能性结合。此外，在A3013Cβ和G3413Cα附近观察到金刚烷胺的〜（15）N标记铵，表明可能与A30羰基形成氢键。需要低温DNP才能观察到ZF-TEDOR光谱中较弱的外部结合位点。这种方法通常适用，特别是对于弱结合的配体，在这种情况下，MAS NMR双极偶联的应用需要低温来淬灭动态交换过程。对于所研究的全质子化样品，我们仅使用4-6 mM的偏振剂TOTAPOL观察到在400 MHz时DNP信号增强了〜10。在600 MHz和DNP条件下，我们测量的药物与蛋白质之间的距离的精确度为0.2？。

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