Tutkus, Marijonas and Akhtar, Parveen and Chmeliov, Jevgenij and Görföl, Fanni and Trinkunas, Gediminas and Lambrev, Petar (2018) Fluorescence Microscopy of Single Liposomes with Incorporated Pigment-Proteins. LANGMUIR, 34 (47). pp. 14410-14418. ISSN 0743-7463
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Abstract
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 singleliposome 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.
| Item Type: | Article |
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| Additional Information: | Funding Agency and Grant Number: Lithuanian-Ukrainian grant [P-LU-18-64]; Hungarian Ministry for National Economy [GINOP-2.3.2-15-2016-00001]; National Research, Development and Innovation Office [NKFIH NN-124904] Funding text: This work was supported by Lithuanian-Ukrainian grant (P-LU-18-64) and by a grant from the Hungarian Ministry for National Economy (GINOP-2.3.2-15-2016-00001) and the National Research, Development and Innovation Office (NKFIH NN-124904). The authors thank D. Rutkauskas for help on setting up the confocal microscope and participation in the initial discussion on the project; M. Vengris for lending hardware and help with the signal acquisition software; and O. Venckus and I. Ciplys for participation in performing several TIRF and confocal microscopy measurements. |
| Uncontrolled Keywords: | MEMBRANES; DYNAMICS; PHOTOSYSTEM-II; Chemistry, Physical; SWITCH; RECONSTITUTION; ANTENNA; EXCITATION-ENERGY; HARVESTING COMPLEX II; Chemistry, Multidisciplinary; MOLECULE SPECTROSCOPY; |
| Subjects: | Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia |
| SWORD Depositor: | MTMT SWORD |
| Depositing User: | MTMT SWORD |
| Date Deposited: | 01 Feb 2019 08:53 |
| Last Modified: | 28 Nov 2019 00:15 |
| URI: | http://real.mtak.hu/id/eprint/90966 |
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