Dynamic Nuclear Polarization Study of Inhibitor Binding to the M218-60 Proton Transporter from Influenza A
Dynamic Nuclear Polarization Study of Inhibitor Binding to the M218-60 Proton Transporter from Influenza A
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DOI：
10.1021/bi400150x
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发表时间：
2013-04-23
期刊：
影响因子：
2.9
通讯作者：
Griffin, Robert G.
中科院分区：
文献类型：
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作者：
Andreas, Loren B.;Barnes, Alexander B.;Griffin, Robert G.
我们演示了使用动态核极化 (DNP) 通过偶极耦合魔角旋转 (MAS) NMR 来阐明配体与膜蛋白的结合。特别是，我们使用室温和低温 DNP 的重偶联实验检测甲型流感质子转运蛋白 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 A30 C-13 β 和 G34 C-13 α 附近观察到 N-15 标记的金刚乙胺铵，表明可能与 A30 羰基存在氢键。需要低温 DNP 来观察 ZF-TEDOR 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 偶极重偶联的应用需要低温来淬灭动态交换过程。对于所研究的完全质子化的样品，我们仅使用 4-6 mM 的极化剂 TOTAPOL 就观察到 DNP 信号在 400 MHz 下增强了类似于 10。在 600 MHz 下并使用 DNP，我们测量了药物和蛋白质之间的距离，精度为 0.2 埃。
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 N-15-labeled ammonium of rimantadine was observed near A30 C-13 beta and G34 C-13 alpha, 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 similar to 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 angstrom.