tetano
Editor, Senior Moderator
Membranes (Basel). 2018 Nov 8;8(4). pii: E103. doi: 10.3390/membranes8040103.
[h=1]Optimization of Detergent-Mediated Reconstitution of Influenza A M2 Protein into Proteoliposomes.[/h] Crouch CH[SUP]1[/SUP], Bost MH[SUP]2[/SUP], Kim TH[SUP]3[/SUP], Green BM[SUP]4[/SUP], Arbuckle DS[SUP]5[/SUP], Grossman CH[SUP]6[/SUP], Howard KP[SUP]7[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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.
[h=4]KEYWORDS:[/h] colorimetric assay; detergent-mediated reconstitution; dynamic light scattering; influenza M2 protein; integral membrane protein; octylglucoside detergent; proteoliposomes
PMID: 30413063 DOI: 10.3390/membranes8040103
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[h=1]Optimization of Detergent-Mediated Reconstitution of Influenza A M2 Protein into Proteoliposomes.[/h] Crouch CH[SUP]1[/SUP], Bost MH[SUP]2[/SUP], Kim TH[SUP]3[/SUP], Green BM[SUP]4[/SUP], Arbuckle DS[SUP]5[/SUP], Grossman CH[SUP]6[/SUP], Howard KP[SUP]7[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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.
[h=4]KEYWORDS:[/h] colorimetric assay; detergent-mediated reconstitution; dynamic light scattering; influenza M2 protein; integral membrane protein; octylglucoside detergent; proteoliposomes
PMID: 30413063 DOI: 10.3390/membranes8040103
Free full text