A new method for the reconstitution of membrane proteins into giant unilamellar vesicles. Export

@article{Girard2004, abstract = {In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy.}, author = {Girard, Philippe and P\'{e}cr\'{e}aux, Jacques and Lenoir, Guillaume and Falson, Pierre and Rigaud, Jean-Louis L. and Bassereau, Patricia}, citeulike-article-id = {2402902}, citeulike-linkout-0 = {http://dx.doi.org/10.1529/biophysj.104.040360}, citeulike-linkout-1 = {http://www.biophysj.org/cgi/content/abstract/87/1/419}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/15240476}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=15240476}, day = {1}, doi = {10.1529/biophysj.104.040360}, issn = {0006-3495}, journal = {Biophysical journal}, keywords = {bacteriorhodopsin, electroformation, vesicle}, month = {July}, number = {1}, pages = {419--429}, posted-at = {2009-11-13 17:17:12}, priority = {2}, title = {A new method for the reconstitution of membrane proteins into giant unilamellar vesicles.}, url = {http://dx.doi.org/10.1529/biophysj.104.040360}, volume = {87}, year = {2004} }

TY - JOUR ID - Girard2004 L3 - citeulike-article-id:2402902 N2 - In this work, we have investigated a new and general method for the reconstitution of membrane proteins into giant unilamellar vesicles (GUVs). We have analyzed systematically the reconstitution of two radically different membrane proteins, the sarcoplasmic reticulum Ca(2+)-ATPase and the H(+) pump bacteriorhodopsin. In a first step, our method involved a detergent-mediated reconstitution of solubilized membrane proteins into proteoliposomes of 0.1-0.2 microm in size. In a second step, these preformed proteoliposomes were partially dried under controlled humidity followed, in a third step, by electroswelling of the partially dried film to give GUVs. The physical characteristics of GUVs were analyzed in terms of morphology, size, and lamellarity using phase-contrast and differential interference contrast microscopy. The reconstitution process was further characterized by analyzing protein incorporation and biological activity. Both membrane proteins could be homogeneously incorporated into GUVs at lipid/protein ratios ranging from 5 to 40 (w/w). After reconstitution, both proteins retained their biological activity as demonstrated by H(+) or Ca(2+) pumping driven by bacteriorhodopsin or Ca(2+)-ATPase, respectively. This constitutes an efficient new method of reconstitution, leading to the production of large unilamellar membrane protein-containing vesicles of more than 20 microm in diameter, which should prove useful for functional and structural studies through the use of optical microscopy, optical tweezers, microelectrodes, or atomic force microscopy. IS - 1 JF - Biophysical journal SN - 0006-3495 EP - 429 TI - A new method for the reconstitution of membrane proteins into giant unilamellar vesicles. VL - 87 SP - 419 KW - bacteriorhodopsin KW - electroformation KW - vesicle AU - Girard, Philippe AU - Pécréaux, Jacques AU - Lenoir, Guillaume AU - Falson, Pierre AU - Rigaud, Jean-Louis AU - Bassereau, Patricia PY - 2004/07/01/ UR - http://dx.doi.org/10.1529/biophysj.104.040360 ER -