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Comparative studies on detergent-assisted apocytochrome b6 reconstitution into liposomal bilayers monitored by Zetasizer instruments.

ABSTRACT: The present paper is a systematic, comparative study on the reconstitution of an apocytochrome b6 purified from a heterologous system using a detergent-free method and reconstitution into liposomes performed using three different detergents: SDS, Triton X-100 and DM, and two methods of detergent removal by dialysis and using Bio-Beads. The product size, its distribution and zeta potential, and other parameters were monitored throughout the process. We found that zeta potential of proteoliposomes is correlated with reconstitution efficiency and, as such, can serve as a quick and convenient quality control for reconstitution experiments. We also advocate using detergent-free protein purification methods as they allow for an unfettered choice of detergent for reconstitution, which is the most crucial factor influencing the final product parameters.

SUBMITTER: Surma MA

PROVIDER: S-EPMC4244035 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Comparative studies on detergent-assisted apocytochrome b6 reconstitution into liposomal bilayers monitored by Zetasizer instruments.

Surma Michał A MA Szczepaniak Andrzej A Króliczewski Jarosław J

PloS one 20141125 11

The present paper is a systematic, comparative study on the reconstitution of an apocytochrome b6 purified from a heterologous system using a detergent-free method and reconstitution into liposomes performed using three different detergents: SDS, Triton X-100 and DM, and two methods of detergent removal by dialysis and using Bio-Beads. The product size, its distribution and zeta potential, and other parameters were monitored throughout the process. We found that zeta potential of proteoliposomes ...[more]

PMID: 25423011

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Project description:Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB(2), and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB(2), here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB(2) were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of (2)H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB(2) in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB(2) reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions.

Dataset Information

Comparative studies on detergent-assisted apocytochrome b6 reconstitution into liposomal bilayers monitored by Zetasizer instruments.

Publications

Comparative studies on detergent-assisted apocytochrome b6 reconstitution into liposomal bilayers monitored by Zetasizer instruments.

Similar Datasets

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