Technical Highlight - March 2014
Short description: Dynamic nuclear polarization is used to enhance the sensitivity of an MAS NMR experiment, allowing detection of inhibitor binding to the M2 proton channel.
Magic angle spinning (MAS) solid-state NMR is becoming an important approach in membrane protein structure studies. Local order and a homogeneous environment in the protein sample, afforded by the 'solid-state' of the lipid membrane environment, are sufficient for structure determination by MAS NMR. In addition, larger biomolecules can be studied without the resonance broadening observed in solution state NMR studies. However, because a lower protein concentration is used, the detected magnetic moments from the 13C and 15N spins are smaller, resulting in decreased resonance sensitivity. Dynamic nuclear polarization (DNP) is a technique for transferring polarization from unpaired electrons to nearby nuclei to enhance the signal-to-noise ratio of the NMR spectra, and has been shown to substantially increase the sensitivity of MAS NMR.
Recently, Griffin and colleagues (PSI MPSbyNMR) used cryogenic DNP in conjunction with MAS NMR to examine the weak binding of the antiviral drug rimantadine (Rmt) to the tetrameric M2 proton channel from influenza A. Prior structural studies using differing M2 constructs identified two distinct binding sites for Rmt and its inhibitory action—a solution NMR study suggested binding at an external site near residues Asp44 and Arg45 of the channel, while crystallographic work indicated binding at an internal site near residues Val27, Ser31 and G34A.
To resolve this controversy, the authors measured the dipolar coupling between uniformly-labeled 13C-labeled M2 and 15N-labeled Rmt reintroduced during MAS by using z-filtered transferred echo double resonance (ZF-TEDOR) that had been enhanced by DNP. The conditions of the DNP-enhanced experiments—low temperature (80–100 K) in the presence of cryoprotectant (60% (v/v) glycerol)—quench the dynamic processes that can interfere with recoupling experiments. Data collected at room temperature showed Rmt binding near Gly34 and Ala30; however, at low temperature using DNP, binding was also observed at the periphery of the protein. These data indicate that the addition of glycerol to the sample before the drug increases the energy barrier for functional drug binding. This and other chemical shift data point to the role of the internal pore site in chemical inhibition.
Using the constraints provided by ZF-TEDOR and prior structural studies, the authors were able to model Rmt binding, with its amine group positioned between Gly34 and Ala30 on one helix of the M2 tetramer. This study demonstrates how DNP can be used to improve MAS NMR experiments to detect weak protein-ligand interactions.
L.B. Andreas et al. Dynamic nuclear polarization study of inhibitor binding to the M2(18–60) proton transporter from influenza A.
Biochemistry. 52, 2774-82 (2013). doi:10.1021/bi400150x