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Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling
Published on: October 21, 2018
13.0K
Determining the amphipol distribution within membrane-protein fibre samples using small-angle neutron scattering
Wanatchaporn Arunmanee1, Richard K Heenan2, Jeremy H Lakey3
1Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
Acta Crystallographica. Section D, Structural Biology
|January 4, 2019
Summary
Free amphipol (APol) polymeric surfactants are essential for maintaining membrane protein stability. Removing free APol causes outer membrane protein F (OmpF) to form filaments, establishing a new equilibrium with free APol.
Area of Science:
- Biochemistry
- Structural Biology
- Materials Science
Background:
- Detergent micelles are crucial for solubilizing membrane proteins, requiring free detergent at critical micellar concentration for equilibrium.
- Amphipol (APol) polymeric surfactants offer an alternative to detergents in membrane protein studies, but the function of free APol is not well understood.
- Previous studies indicated that removing free APol leads to outer membrane protein F (OmpF) forming filaments, though APol distribution remained unresolved.
Purpose of the Study:
- To elucidate the role of free amphipol (APol) in the structural dynamics of membrane protein complexes.
- To investigate the equilibrium established between protein-amphipol filaments and free amphipol after depletion.
- To characterize the distribution and structure of amphipol and membrane proteins in solution.
Main Methods:
- Small-angle neutron scattering (SANS) with isotope contrast matching was employed to differentiate and quantify membrane protein and amphipol distributions.
- Analysis of monodisperse outer membrane protein F (OmpF) complexes subjected to free amphipol removal.
- Microscopy techniques were used to complement scattering data.
Main Results:
- Small-angle neutron scattering revealed a dynamic equilibrium between outer membrane protein F (OmpF)-amphipol (APol) filaments and a pool of newly liberated free APol after initial depletion.
- The OmpF-APol filaments were characterized by OmpF proteins encircled by a belt of APol.
- Free amphipol existed as oblate spheroid micelles, with no evidence of long-range order in the filaments, suggesting structural flexibility.
Conclusions:
- Free amphipol plays a critical role in maintaining the structural integrity and solubility of membrane proteins like OmpF.
- The formation of OmpF-APol filaments and the subsequent release of free amphipol demonstrate a dynamic self-assembly and equilibrium process.
- Amphipols, particularly the free form, are essential for stabilizing membrane proteins in solution, offering insights into protein-surfactant interactions.
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