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Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis
Published on: October 28, 2016
Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide
Patricia E Nigg1, Jovan Pavlovic2
1Institute for Medical Virology, University of Zurich; Friedrich Miescher Institute for Biomedical Research.
Abstract:
The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.
Insights
This study presents a simple protocol to assess the oligomeric state of MxA, an antiviral protein. The method helps correlate MxA oligomerization with its antiviral activity, clarifying its mechanism of action.
Area of Science:
- Biochemistry
- Molecular Biology
- Virology
Background:
- Protein oligomerization is vital for cellular function.
- MxA is an interferon-induced GTPase with broad antiviral activity.
- The role of MxA oligomerization in its antiviral function remains debated.
Purpose of the Study:
- To develop a straightforward protocol for assessing MxA oligomeric state.
- To correlate MxA oligomerization with its antiviral activity.
- To investigate MxA interface mutants' oligomeric states and functions.
Main Methods:
- Non-denaturing polyacrylamide gel electrophoresis (PAGE) coupled with Western blot analysis.
- Utilizing specific detergents to maintain MxA structure and enzymatic activity.
- Irreversible protection of cysteine thiols using iodoacetamide to prevent artifactual interactions.
Main Results:
- A reliable protocol was established to determine the oligomeric state of MxA in human cell lines.
- The method allows for the assessment of endogenously and ectopically expressed MxA.
- The protocol facilitates direct correlation between MxA oligomeric state and antiviral activity.
Conclusions:
- The developed protocol simplifies the assessment of MxA oligomeric status.
- This technique enables a direct link between MxA oligomerization and its antiviral efficacy.
- The findings contribute to understanding MxA's mechanism of action in antiviral defense.
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