Dynamic Nuclear Polarization Study of Inhibitor Binding to the M218鈥?0 Proton Transporter from Influenza A

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• 作者：Loren B. Andreas ; Alexander B. Barnes ; Bj枚rn Corzilius ; James J. Chou ; Eric A. Miller ; Marc Caporini ; Melanie Rosay ; Robert G. Griffin
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：April 23, 2013
• 年：2013
• 卷：52
• 期：16
• 页码：2774-2782
• 全文大小：415K
• 年卷期：v.52,no.16(April 23, 2013)
• ISSN：1520-4995

文摘

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鈥?0 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 ¹⁵N-labeled ammonium of rimantadine was observed near A30 ¹³C尾 and G34 ¹³C伪, 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鈥? 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 脜.