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Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis
Published on: October 28, 2016
Polar residues in transmembrane helices can decrease electrophoretic mobility in polyacrylamide gels without causing
William F Walkenhorst1, Mikhail Merzlyakov, Kalina Hristova
1Department of Chemistry, Loyola University, New Orleans, LA 70118, USA.
Biochimica Et Biophysica Acta
|March 7, 2009
Summary
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) can inaccurately suggest membrane protein dimerization. Differential peptide-detergent interactions, not dimerization, cause altered migration in SDS-PAGE, highlighting the need for multiple study techniques.
Area of Science:
- Biochemistry
- Biophysics
- Membrane Protein Research
Background:
- Studying transmembrane helix interactions and self-assembly is challenging.
- Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a common method for assessing peptide interactions in detergent solutions.
- SDS-PAGE is often used as a membrane mimetic system for studying membrane protein folding and assembly.
Purpose of the Study:
- To investigate the reliability of SDS-PAGE for studying membrane protein folding and assembly.
- To examine the potential for artifacts in SDS-PAGE experiments when studying peptides in detergent solutions.
- To elucidate the factors influencing the electrophoretic mobility of peptides in SDS-PAGE.
Main Methods:
- SDS-PAGE to assess peptide migration and apparent molecular weight.
- Forster Resonance Energy Transfer (FRET) to detect peptide interactions in solution and within gels.
- Analysis of peptide-detergent interactions.
Main Results:
- A polar asparagine residue caused a peptide to migrate with an apparent molecular weight double its true weight on SDS-PAGE, suggesting dimerization.
- FRET analysis detected no actual peptide-peptide interactions in SDS solutions or within the gel.
- Evidence suggests that differential interactions between the peptide and detergent, not dimerization, caused the observed mobility shifts.
Conclusions:
- SDS-PAGE can produce artifactual results regarding membrane protein dimerization.
- Differential peptide-detergent interactions are a critical factor influencing electrophoretic mobility in SDS-PAGE.
- Multiple independent techniques are essential for accurate studies of membrane protein folding and assembly, preferably in lipid membranes over detergents.
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