Fluorescence Microscopy of Single Liposomes with Incorporated Pigment-Proteins
Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio i...
| Publié dans: | Langmuir : the ACS journal of surfaces and colloids. - 1985. - 34(2018), 47 vom: 27. Nov., Seite 14410-14418 |
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| Auteur principal: | |
| Autres auteurs: | , , , , , |
| Format: | Article en ligne |
| Langue: | English |
| Publié: |
2018
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| Accès à la collection: | Langmuir : the ACS journal of surfaces and colloids |
| Sujets: | Journal Article Research Support, Non-U.S. Gov't Liposomes Photosystem II Protein Complex Proteolipids proteoliposomes |
| Résumé: | Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the single-liposome level and for the first time apply it to study photosynthetic pigment-protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment |
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| Description: | Date Completed 04.02.2019 Date Revised 09.01.2024 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1520-5827 |
| DOI: | 10.1021/acs.langmuir.8b02307 |