PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons

Related Articles
Automating NMR Structures
April 2015
Protein Folding and Misfolding: A TRiC-ster that Follows the Rules
March 2015
Virology: Making Sensitive Magic
March 2014
Microbiome: Solid-State NMR, Crystallized
September 2013
Membrane Proteome: Making DNA Nanotubes for NMR Structure Determination
August 2013
Protein-Nucleic Acid Interaction: Inhibition Through Allostery
July 2013
Cell-Cell Interaction: Magic Structure from Microcrystals
March 2013
Membrane Proteome: Soft Sampling
December 2012
Membrane Proteome: Specific vs. Non-specific weak interactions
November 2012
Automatic NMR
September 2012
NMR structure test
September 2012
To structure, faster
August 2012
S is for solubility
June 2012
Blind faith
April 2012
Follow the RNA leader
December 2011
Making invisible proteins visible
October 2011
A fragmented approach to membrane protein structures
September 2011
Molecular replacement by magnetic resonance
August 2011
Solutions in the solution
June 2011
No more labeled lipids
May 2011
Capsid assembly in motion
April 2011
NMR challenges current protein hydration dogma
March 2011
Solving homodimeric structures with NMR
November 2010
CASD-NMR: assessing automated structure determination by NMR
June 2010
Peptidoglycan binding: Calcium-free killing
June 2010
Removing the NMR bottleneck
April 2010
NMR has its wiki way
March 2010
Extremely salty
February 2010
The future of NMR
September 2009
Tips for crystallizing membrane proteins
June 2009
Faster solid-state NMR
May 2009
Powerful NMR
April 2009
Activating BAX
December 2008

Virology: Making Sensitive Magic

SBKB [doi:10.1038/sbkb.2012.191]
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.

Structural model of Rmt interacting with the internal pore site of M2. M2 is in cyan and Rmt is in green. Reprinted with permission from the American Chemical Society. 1

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.

Michelle Montoya


  1. 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

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health