Reconstitution of electrochromically active pigment-protein complexes from Rhodobacter sphaeroides into liposomes (original) (raw)
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Carotenoids in Liposomes: Photodegradation, Excited State Lifetimes, and Energy Transfer
The Journal of Physical Chemistry B, 2000
DMPC (dimyristoyl-L-R-phosphatidylcholine) liposomes are used as artificial photosynthetic media to study the behavior of carotenoids. 8′-Apo--caroten-8′-al (I) and -carotene (II) degrade faster under irradiation in DMPC liposomes than in organic solvents, which is possibly because vibrational deactivation of carotenoid excited states is less efficient in rigid lipid membranes. The lifetime of the first excited singlet state (S 1 ) of I in DMPC liposomes is 27.2 ps, very close to that in 3-methylpentane (26.4 ps), but longer than its lifetime in EtOH (17.1 ps) or CH 2 Cl 2 (14.1 ps). The lifetime of the S 1 state of I in DMPC liposomes is as expected for an alkane environment. The lifetime of the S 1 state of II in DMPC liposomes is 10.3 ps, very close to its lifetimes in 3-methylpentane (8.1 ps), EtOH (9.2 ps), and CH 2 Cl 2 (8.5 ps). This independence of the S 1 state lifetime of II from the matrix agrees with earlier conclusions. Carotenoid I can suppress the photodegradation of chlorophyll a (Chl a) in liposomes, which shows the protection role of I on Chl a under strong irradiation. In liposomes, Chl a fluorescence quenching by I is observed when using either the Q y band or the Soret band of Chl a as the excitation line.
Fluorescence Microscopy of Single Liposomes with Incorporated Pigment–Proteins
Langmuir, 2018
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 labelling, 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 non-reconstituted 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 native-like environment.
Selective Electrochemical Bleaching of the Outer Leaflet of Fluorescently Labelled Giant Liposomes
Chemistry (Weinheim an der Bergstrasse, Germany), 2017
Electrochemistry and confocal fluorescence microscopy were successfully combined to selectively bleach and monitor the fluorescence of NBD-labelled phospholipids of giant liposomes. Three types of giant unilamellar vesicles have been investigated, the fluorescent phospholipids being localized either mainly on their outer-, inner-, or both inner/outer leaflets. We established that only the fluorescent lipids incorporated in the outer leaflet of the vesicles underwent electrochemical bleaching upon reduction. The relative fluorescence intensity decay was quantified all along the electrochemical extinction through an original Fluorescence Loss In Electrobleaching (FLIE) assay. As expected, the re-organization of the fluorescent phospholipids followed diffusion-driven dynamics. This was also evidenced by comparison with Fluorescence Loss In Photobleaching (FLIP) and the corresponding numerical model. The value of the lateral diffusion coefficient of phospholipids was found to be similar...
A Stability Test of Liposome Preparations Using Steady-State Fluorescent Measurements
Drug Delivery, 2001
The stability of liposome preparations under the action of the nonionic detergent Triton X-100 was measured using the fluorescent molecular probe octadecylrhodamine B (R18). The probe inserted in the lipid bilayer shows a self-quenched fluorescence and the degree of quenching depends both on the probe concentration and the phase state of the lipid membrane. The addition of detergent to the liposomes produces a steep decrease in self-quenching caused by dilution of the probe in the bilayer. The curves of steady-state fluorescence intensity show an abrupt change in slope that corresponds to the point at which liposomes break down into lipid-detergent mixed entities that are different from the earlier liposome-monodisperse population. The lytic process was followed in parallel by dynamic light scattering (DLS), and the analysis of the DLS results agree with the interpretation of the fluorescence measurements. The probe R18 therefore is a useful marker to test the stability of liposome ...
Determination of liposome size: A tool for protein reconstitution
Analytical Biochemistry, 2005
Reconstitution of proteins into liposomes is a widespread approach to analyzing their biological function. Many protocols exist for this procedure and for the subsequent analysis of proteins. Here, we establish a procedure for preparation and analysis of liposomes with a lipid composition reflecting the outer envelope of chloroplasts. First, the stability of the liposomes in different buffer systems was investigated to provide information for the storage of the reconstituted system. Then, the size of the liposomes created by filtration through a polycarbonate filter dependent on the lipid composition was analyzed. Subsequently, solubilization of the liposomes composed of lipids with the outer envelope composition by dodecylmaltoside and octylglucoside as a preceding step of reconstitution was studied. Finally, we developed a straightforward method to determine the size of liposomes by absorption spectroscopy. The described setup allows the construction of reconstitution protocols, including the final determination of the liposome size.
The Journal of Cell Biology, 1985
Chlorophyll a/b light-harvesting complexes (chl a/b LHC) and photosystem II (PSII) cores were isolated from an octyl glucoside-containing sucrose gradient after solubilization of barley thylakoid membranes with Triton X-100 and octyl glucoside. No cation precipitation step was necessary to collect the chl a/b LHC. PAGE under mildly denaturing and fully denaturing conditions showed that the chl a/b LHC fraction contained chlorophyll-protein complexes CP27, CP29, and CP64. The PSII core material contained CP43 and CP47, and little contamination by other nonpigmented polypeptides. Freeze-fracture electron microscopy of the chl a/b LHC after reconstitution into digalactosyldiglyceride (DG) or phosphatidylcholine (PC) vesicles showed that the protein particles (approximately 7.5 +/- 1.6 nm) were approximately 99 and 90% randomly dispersed, respectively, in the liposomes. Addition of Mg++ produced particle aggregation and membrane adhesion in chl a/b LHC-DG liposomes in a manner analogous...
Drug Delivery, 1998
Cationic liposomes are being increasingly studied as delivery vehicles for bioactive agents such as DNA and other polynucleotides. The mechanism of interaction of DNA with liposomes and the organization of these interacting structures during and after the interaction are still poorly understood. Nucleic acids are known to induce aggregation and size enlargement of liposomes. In the case of phosphatidylcholine (PC) vesicles, these processes depend on the presence and concentration of divalent metal cations and the amount of cholesterol in the liposomes. In this study, anionic small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) composed of dicetylphosphate (DCP):PC:cholesterol at 2:7:1 molar ratios were prepared and incubated with the DNA (from wheat) and CaZ+ (50 mM) at 25°C with the aim of transferring the genetic material into the liposomes by inducing fusion of liposome-liposome aggregates created in the presence, and with the help, of DNA, The organization and the nature of the resultant Uposome-DNA-Ca'+ complexes were investigated by scanning tunneling microscopy (STM) and fluorescence microscopy. Observations of complexes with similar appearances with both SUV and MLV, as shown by two quite different microscopic approaches, prove that the resultant forms are real and not artifacts of the methodology used. At this stage it is not clear whether the detected complexes represent an intermediate state before fusion of liposomes which will lead to engulfing of the genomic material by the fused liposomes, or the final form. In either case the structures consisting of some adhered or semifused liposomes bearing the nucleic acid seem to be candidates as vehicles for in-vitro and in-vivo transfection.