skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Influenza AM2 Channel Oligomerization Is Sensitive to Its Chemical Environment
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 and other techniques to characterize the oligomeric state of both the full-length and transmembrane (TM) domain of 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 the local chemical environment. Native mass spectrometry of AM2 in nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled the measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 can form a wider range of oligomeric states than previously thought possible, which may provide new potential mechanisms of influenza pathology and pharmacology.  more » « less
Award ID(s):
2018942
PAR ID:
10334588
Author(s) / Creator(s):
; ; ; ;
Editor(s):
Sweedler, J. V.
Date Published:
Journal Name:
Analytical chemistry
Volume:
93
Issue:
48
ISSN:
0003-2700
Page Range / eLocation ID:
16273-16281
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; (, The Analyst)
    Native electrospray mass spectrometry is a powerful method for determining the native stoichiometry of many polydisperse multi-subunit biological complexes, including multi-subunit protein complexes and lipid-bound transmembrane proteins. However, when polydispersity results from incorporation of multiple copies of two or more different subunits, it can be difficult to analyze subunit stoichiometry using conventional mass spectrometry analysis methods, especially when m / z distributions for different charge states overlap in the mass spectrum. It was recently demonstrated by Marty and co-workers (K. K. Hoi, et al. , Anal. Chem. , 2016, 88 , 6199–6204) that Fourier Transform (FT)-based methods can determine the bulk average lipid composition of protein-lipid Nanodiscs assembled with two different lipids, but a detailed statistical description of the composition of more general polydisperse two-subunit populations is still difficult to achieve. This results from the vast number of ways in which the two types of subunit can be distributed within the analyte ensemble. Here, we present a theoretical description of three common classes of heterogeneity for mixed-subunit analytes and demonstrate how to differentiate and analyze them using mass spectrometry and FT methods. First, we first describe FT-based analysis of mass spectra corresponding to simple superpositions, convolutions, and multinomial distributions for two or more different subunit types using model data sets. We then apply these principles with real samples, including mixtures of single-lipid Nanodiscs in the same solution (superposition), mixed-lipid Nanodiscs and copolymers (convolutions), and isotope distribution for ubiquitin (multinomial distribution). This classification scheme and the FT method used to study these analyte classes should be broadly useful in mass spectrometry as well as other techniques where overlapping, periodic signals arising from analyte mixtures are common. 
    more » « less
  2. ; ; ; ; (, Chemical Communications)
    null (Ed.)
    In the study of membrane proteins and antimicrobial peptides, nanodiscs have emerged as a valuable membrane mimetic to solubilze these molecules in a lipid bilayer. We present the structural characterization of nanodiscs using native mass spectrometry and surface-induced dissociation, which are powerful tools in structural biology. 
    more » « less
  3. ; ; ; (, Current Pharmaceutical Design)
    null (Ed.)
    Background: The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomersbut that the oligomers also may modulate the receptor function provides a promising avenue in the area ofdrug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacywhile reducing side effects. In this regard, determining the various oligomeric configurations and geometricsub-states of a membrane receptor is of utmost importance. Methods: In this report, we have reviewed two techniques that have proven to be valuable in monitoring thequaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescenceintensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiencyare analyzed using theoretical models of various quaternary structures to determine the geometry andstoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensitiesare analyzed to reveal quantitative information on the size and stability of protein oligomers. Results: We demonstrate the application of these techniques to a number of different fluorescence-based studiesof cells expressing fluorescently labeled membrane receptors, both in the presence and absence of variousligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regardingthe quaternary structure receptors form, as well as FIF and FRET for determining the relative abundanceof different-sized oligomers when an equilibrium forms between such structures. Conclusion: FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers,which are of great benefit to structure‐based drug design. 
    more » « less
  4. ; ; ; ; ; (, Analytical Chemistry)
    Analyzing lipid assemblies, including liposomes and extracellular vesicles (EVs), is challenging due to their size, diverse composition, and tendency to aggregate. Such vesicles form with a simple phospholipid bilayer membrane, and they play important roles in drug discovery and delivery. The use of mass spectrometry (MS) allows for broad analysis of lipids from different classes; however, their release from the higher order structural aggregates is typically achieved by chemical means. Mechanical disruption by high frequency surface acoustic waves (SAW) is presented as an appealing alternative to preparing lipid vesicles for MS sampling. In this work, SAWs used to disrupt liposomes allow for the direct analysis of their constituent lipids by employing SAW nebulization with corona discharge (CD) ionization. We explore the effects of duration, frequency, and incorporation of nonpolar lipids, including cholesterol, on the SAW’s ability to disrupt the liposome. We also report on the successful MS analysis of liposome-derived lipids along with cytochrome C in solution, thus demonstrating applications to aqueous samples and native MS conditions. 
    more » « less
  5. ; ; ; (, Journal of Computational Chemistry)
    Nanodiscs are discoidal protein–lipid complexes that have wide applications in membrane protein studies. Modeling and simulation of nanodiscs are challenging due to the absence of structures of many membrane scaffold proteins (MSPs) that wrap around the membrane bilayer. We have developed CHARMM‐GUINanodisc Builder(http://www.charmm-gui.org/input/nanodisc) to facilitate the setup of nanodisc simulation systems by modeling the MSPs with defined size and known structural features. A total of 11 different nanodiscs with a diameter from 80 to 180 Å are made available in both the all‐atom CHARMM and two coarse‐grained (PACE and Martini) force fields. The usage of theNanodisc Builderis demonstrated with various simulation systems. The structures and dynamics of proteins and lipids in these systems were analyzed, showing similar behaviors to those from previous all‐atom and coarse‐grained nanodisc simulations. We expect theNanodisc Builderto be a convenient and reliable tool for modeling and simulation of nanodisc systems. © 2019 Wiley Periodicals, Inc. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email