PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons

Related Articles
Protein Folding and Misfolding: It's the Journey, Not the Destination
March 2015
CCR5 and HIV Infection
January 2015
HIV/AIDS: Pre-fusion Env Exposed
January 2015
HIV/AIDS: Slide to Enter
January 2015
Updating ModBase
January 2015
Power in Numbers
August 2014
Quorum Sensing: A Groovy New Component
August 2014
Bacterial CDI Toxins
June 2014
Immunity: One Antibody to Rule Them All
June 2014
Virology: A Bat Influenza Hemagglutinin
March 2014
Virology: Making Sensitive Magic
March 2014
Virology: Visualizing Cyanophage Assembly
March 2014
Virology: Zeroing in on HBV Egress
March 2014
March 2014
Cas4 Nuclease and Bacterial Immunity
February 2014
Microbial Pathogenesis: A GNAT from Pseudomonas
February 2014
Microbial Pathogenesis: Targeting Drug Resistance in Mycobacterium tuberculosis
February 2014
Microbiome: The Dynamics of Infection
September 2013
Membrane Proteome: A Funnel-like Viroporin
August 2013
Infectious Diseases: A Pathogen Ubiquitin Ligase
May 2013
Infectious Diseases: A Shared Syringe
May 2013
Infectious Diseases: Determining the Essential Structome
May 2013
Infectious Diseases: Targeting Meningitis
May 2013
NDM-1 and Antibiotics
May 2013
Bacterial Hemophores
January 2013
Microbial Pathogenesis: Computational Epitope Prediction
January 2013
Microbial Pathogenesis: Influenza Inhibitor Screen
January 2013
Microbial Pathogenesis: Measles Virus Attachment
January 2013
Microbial Pathogenesis: NEAT Iron
January 2013
Membrane Proteome: Sphingolipid Synthesis Selectivity
December 2012
A signal sensing switch
September 2012
Gauging needle structure
July 2012
Anthrax Stealth Siderophores
June 2012
A Pseudomonas L-serine dehydrogenase
May 2012
Pilus Assembly Protein TadZ
April 2012
Making Lipopolysaccharide
January 2012
Superbugs and Antibiotic Resistance
December 2011
A change to resistance
November 2011
An effective and cooperative dimer
November 2011
The Perils of Protein Secretion
November 2011
Bacterial Armor
October 2011
Breaking down the defenses
September 2011
Moving some metal
August 2011
Capsid assembly in motion
April 2011
Know thy enemy … structurally
October 2010
Treating sleeping sickness
May 2010
Bacterial spore kinase
April 2010
Hemolysin BL
January 2010
Unusual cell division
October 2009
Anthrax evasion tactics
September 2009
Toxin-antitoxin VapBC-5
September 2009
Antibiotic target
August 2009
July 2009
Tackling influenza
June 2009
You look familiar: the Type VI secretion system
June 2009
Unique SARS
April 2009
Anthrax stealth molecule
March 2009
A new class of bacterial E3 ubiquitination enzymes
January 2009
Antiviral evasion
October 2008
SARS connections
September 2008
SARS Coronavirus Nonstructural Protein 1
June 2008

Research Themes Infectious diseases

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