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Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics
Published on: February 18, 2022
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Mass Photometry of Membrane Proteins
Anna Olerinyova1, Adar Sonn-Segev1, Joseph Gault1
1Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.
Chem
|January 29, 2021
Summary
Mass photometry (MP) offers a novel method for studying integral membrane proteins (IMPs) in lipid-like environments. This technique characterizes particle size and purity, revealing new insights into membrane protein structure and interactions.
Area of Science:
- Biophysics
- Structural Biology
- Membrane Protein Research
Background:
- Integral membrane proteins (IMPs) are crucial for cellular functions but difficult to study due to their complex environment.
- Existing methods for IMP purification and analysis often alter their native structure or introduce artifacts.
- Single-particle analysis techniques are needed to accurately characterize IMPs in near-native states.
Purpose of the Study:
- To investigate the utility of mass photometry (MP) for analyzing IMPs and membrane-mimetic systems at the single-particle level.
- To assess MP's capability in characterizing particle size, sample purity, and heterogeneity of various membrane-mimetic systems.
- To evaluate MP's potential for high-resolution structural studies and interaction analysis of IMPs.
Main Methods:
- Application of mass photometry (MP) to amphipathic vehicles (detergents, amphipols) and nanodiscs (lipid and native).
- Characterization of particle size, sample purity, and heterogeneity using MP.
- Adaptation of cryogenic electron microscopy (Cryo-EM) methods to eliminate detergent background for enhanced MP analysis.
- Purification and analysis of the potassium channel KcsA using native styrene-maleic acid nanodiscs and lipid nanodiscs.
Main Results:
- MP successfully characterized particle size, purity, and heterogeneity in detergent, amphipol, lipid nanodisc, and native nanodisc systems.
- Elimination of detergent background enabled high-resolution MP studies of membrane protein structure and interactions.
- Analysis of KcsA channel in native nanodiscs indicated a dimer of tetramers, contrasting with detergent-purified results.
- MP differentiated between functional and non-functional lipid nanodisc assemblies and identified key formation factors.
Conclusions:
- Mass photometry is a powerful tool for single-particle analysis of integral membrane proteins and their associated systems.
- MP provides accurate characterization of membrane mimetics, aiding in the selection of optimal systems for IMP studies.
- MP offers a complementary approach to Cryo-EM, enabling high-resolution structural insights and revealing discrepancies in protein oligomerization states based on purification methods.
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