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Membranes 2018, 8(4), 103; https://doi.org/10.3390/membranes8040103

Optimization of Detergent-Mediated Reconstitution of Influenza A M2 Protein into Proteoliposomes

1
Department of Physics & Astronomy, Swarthmore College, Swarthmore, PA 19081, USA
2
Department of Chemistry & Biochemistry, Swarthmore College, Swarthmore, PA 19081, USA
Current Address: Graduate Program in Molecular Biophysics and Medical Scientist Training Program, University of Texas-Southwestern Medical School, Dallas, TX 75235, USA.
Current Address: Brava Home, Inc., Redwood City, CA 94063, USA.
*
Author to whom correspondence should be addressed.
Received: 26 September 2018 / Revised: 27 October 2018 / Accepted: 3 November 2018 / Published: 8 November 2018
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Abstract

We report the optimization of detergent-mediated reconstitution of an integral membrane-bound protein, full-length influenza M2 protein, by direct insertion into detergent-saturated liposomes. Detergent-mediated reconstitution is an important method for preparing proteoliposomes for studying membrane proteins, and must be optimized for each combination of protein and membrane constituents used. The purpose of the reconstitution was to prepare samples for site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) studies. Our goals in optimizing the protocol were to minimize the amount of detergent used, reduce overall proteoliposome preparation time, and confirm the removal of all detergent. The liposomes were comprised of (1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), and the detergent octylglucoside (OG) was used for reconstitution. Rigorous physical characterization was applied to optimize each step of the reconstitution process. We used dynamic light scattering (DLS) to determine the amount of OG needed to saturate the preformed liposomes. During detergent removal by absorption with Bio-Beads, we quantified the detergent concentration by means of a colorimetric assay, thereby determining the number of Bio-Bead additions needed to remove all detergent from the final proteoliposomes. We found that the overnight Bio-Bead incubation used in previously published protocols can be omitted, reducing the time needed for reconstitution. We also monitored the size distribution of the proteoliposomes with DLS, confirming that the size distribution remains essentially constant throughout the reconstitution process. View Full-Text
Keywords: detergent-mediated reconstitution; integral membrane protein; influenza M2 protein; proteoliposomes; octylglucoside detergent; dynamic light scattering; colorimetric assay detergent-mediated reconstitution; integral membrane protein; influenza M2 protein; proteoliposomes; octylglucoside detergent; dynamic light scattering; colorimetric assay
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Crouch, C.H.; Bost, M.H.; Kim, T.H.; Green, B.M.; Arbuckle, D.S.; Grossman, C.H.; Howard, K.P. Optimization of Detergent-Mediated Reconstitution of Influenza A M2 Protein into Proteoliposomes. Membranes 2018, 8, 103.

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