scholarly journals Influenza A M2 Channel Oligomerization is Sensitive to its Chemical Environment

2021 ◽  
Author(s):  
Julia A Townsend ◽  
Henry M Sanders ◽  
Amber D Rolland ◽  
James S Prell ◽  
Jun Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Targets ◽  
Influenza A ◽  
Matrix Protein ◽  
Antiviral Drug ◽  
Native Mass Spectrometry ◽  
Chemical Environment ◽  
Size Exclusion ◽  
Oligomeric State ◽  
Exclusion Chromatography

Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry, ion mobility spectrometry, and size-exclusion chromatography to characterize the oligomeric state of full-length AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by its local chemical environment. The monodisperse tetramer was only observed in select conditions when the antiviral drug, amantadine, was added. Native mass spectrometry of AM2 in lipid nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 forms a wider range of oligomeric states than previously thought possible, which provides new potential mechanisms of influenza pathology and pharmacology.

scholarly journals Characterization of trimethoprim resistant E. coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates

2017 ◽  
Vol 8 (5) ◽  
pp. 4062-4072 ◽  
Author(s):  
Michael Cammarata ◽  
Ross Thyer ◽  
Michael Lombardo ◽  
Amy Anderson ◽  
Dennis Wright ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Dihydrofolate Reductase ◽  
Native Mass Spectrometry ◽  
Size Exclusion ◽  
E Coli ◽  
Exclusion Chromatography

Native mass spectrometry, size exclusion chromatography, and kinetic assays were employed to study trimethoprim resistance in E. coli caused by mutations P21L and W30R of dihydrofolate reductase.

scholarly journals Extending native mass spectrometry approaches to integral membrane proteins

2015 ◽  
Vol 396 (9-10) ◽  
pp. 991-1002 ◽  
Author(s):  
Albert Konijnenberg ◽  
Jeroen F. van Dyck ◽  
Lyn L. Kailing ◽  
Frank Sobott
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Gas Phase ◽  
Conformational Changes ◽  
Drug Targets ◽  
Native Mass Spectrometry ◽  
Lipid Binding ◽  
Drug Binding ◽  
Integral Membrane Proteins ◽  
Oligomeric State

Abstract Recent developments in native mass spectrometry and ion mobility have made it possible to analyze the composition and structure of membrane protein complexes in the gas-phase. In this short review we discuss the experimental strategies that allow to elucidate aspects of the dynamic structure of these important drug targets, such as the structural effects of lipid binding or detection of co-populated conformational and assembly states during gating on an ion channel. As native mass spectrometry relies on nano-electrospray of natively reconstituted proteins, a number of commonly used lipid- and detergent-based reconstitution systems have been evaluated for their compatibility with this approach, and parameters for the release of intact, native-like folded membrane proteins studied in the gas-phase. The strategy thus developed can be employed for the investigation of the subunit composition and stoichiometry, oligomeric state, conformational changes, and lipid and drug binding of integral membrane proteins.

scholarly journals Toward Automation of Collision-Induced Unfolding Experiments through Online Size Exclusion Chromatography Coupled to Native Mass Spectrometry

2020 ◽  
Vol 92 (19) ◽  
pp. 12900-12908 ◽  
Author(s):  
Evolène Deslignière ◽  
Anthony Ehkirch ◽  
Thomas Botzanowski ◽  
Alain Beck ◽  
Oscar Hernandez-Alba ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Native Mass Spectrometry ◽  
Size Exclusion ◽  
Collision Induced Unfolding ◽  
Exclusion Chromatography

scholarly journals Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry

mAbs ◽  
2015 ◽  
Vol 8 (2) ◽  
pp. 331-339 ◽  
Author(s):  
Markus Haberger ◽  
Michael Leiss ◽  
Anna-Katharina Heidenreich ◽  
Oxana Pester ◽  
Georg Hafenmair ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Native Mass Spectrometry ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Rapid Characterization

scholarly journals Probing Protein Denaturation during Size-Exclusion Chromatography Using Native Mass Spectrometry

2020 ◽  
Vol 92 (6) ◽  
pp. 4292-4300 ◽  
Author(s):  
Iro K. Ventouri ◽  
Daniel B. A. Malheiro ◽  
Robert L. C. Voeten ◽  
Sander Kok ◽  
Maarten Honing ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Protein Denaturation ◽  
Native Mass Spectrometry ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals DNA G-quadruplexes for native mass spectrometry in potassium: a database of validated structures in electrospray-compatible conditions

2021 ◽  
Author(s):  
Anirban Ghosh ◽  
Eric Largy ◽  
Valérie Gabelica
Keyword(s):  
Mass Spectrometry ◽  
Drug Targets ◽  
Native Mass Spectrometry ◽  
Drug Binding ◽  
Quadruplex Dna ◽  
Dna Structures ◽  
Nmr Structures ◽  
G Quadruplex ◽  
Screening Assays

Abstract G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually ∼100 mM) potassium, and report here their circular dichroism (CD), melting temperature (Tm), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM trimethylammonium acetate. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).

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