ABSTRACT
Using colloidal particles as models to understand processes on a smaller scale is a precious approach. Compared to molecules, particles are less defined, but their architecture can be more complex and so is their long-range interaction. One can observe phenomena that are unknown or much more difficult to realize on the molecular level. The current paper focuses on particle-based surfactants and reports on numerous unexpected properties. The main goal is creating an amphiphilic system with responsiveness in surface activity and associated self-organization phenomena depending on applying an external trigger, preferably a physical field. A key step is the creation of a Janus-type particle characterized by two types of dipoles (electric and magnetic) which geometrically stand orthogonal to each other. In a field, one can control which contribution and direction dominate the interparticle interactions. As a result, one can drastically change the system's properties. The features of ferrite-core organosilica-shell particles with grain-like morphology modified by click chemistry are studied in response to spatially isotropic and anisotropic triggers. A highly unusual aggregation-dissolution-reaggregation sequence w as discovered. Using a magnetic field, one can even switch off the amphiphilic properties and use this for the field-triggered breaking of multiphase systems such as emulsions.
ABSTRACT
There is no theoretical limit in using molecular networks to harvest diffusive sun photons on large areas and funnel them onto much smaller areas of highly efficient but also precious energy-converting materials. The most effective concept reported so far is based on a pool of randomly oriented, light-harvesting donor molecules that funnel all excitation quanta by ultrafast energy transfer to individual light-redirecting acceptor molecules oriented parallel to the energy converters. However, the best practical light-harvesting system could only be discovered by empirical screening of molecules that either align or not within stretched polymers and the maximum absorption wavelength of the empirical system was far away from the solar maximum. No molecular property was known explaining why certain molecules would align very effectively whereas similar molecules did not. Here, we first explore what molecular properties are responsible for a molecule to be aligned. We found a parameter derived directly from the molecular structure with a high predictive power for the alignability. In addition, we found a set of ultrafast funneling molecules that harvest three times more energy in the solar's spectrum peak for GaInP photovoltaics. A detailed study on the ultrafast dipole moment reorientation dynamics demonstrates that refocusing of the diffusive light is based on â¼15-ps initial dipole moment depolarization followed by â¼50-ps repolarization into desired directions. This provides a detailed understanding of the molecular depolarization/repolarization processes responsible for refocusing diffusively scattered photons without violating the second law of thermodynamics.
ABSTRACT
Transitions into the first excited state of carotenoids, Car S1, are optically forbidden in conventional one-photon excitation (OPE) but are possible via two-photon excitation (TPE). This can be used to quantify the amount of Car S1 to Chlorophyll (Chl) energy transfer in pigment-protein complexes and plants by observing the chlorophyll fluorescence intensity after TPE in comparison to the intensity observed after direct chlorophyll OPE. A parameter, ΦCoupling Car S1-Chl, can be derived that directly reflects relative differences or changes in the Car S1 â Chl energy transfer of different pigment-protein complexes and even living plants. However, very careful calibrations are necessary to ensure similar OPE and TPE excitation probabilities and transition energies. In plants, the exact same sample spot must be observed at the same time. All this is experimentally quite demanding. ΦCoupling Car S1-Chl also corrects intrinsically for direct chlorophyll TPE caused by larger chlorophyll excesses in the complexes, but recently it turned out that in certain TPE wavelengths ranges, its contribution can be quite large. Fortunately, this finding opens also the possibility of determining ΦCoupling Car S1-Chl in a much easier way by directly comparing values in TPE spectra observed at wavelengths that are either more dominated by Cars or Chls. This avoids tedious comparisons of OPE and TPE experiments and potentially allows measurement at even only two TPE wavelengths. Here, we explored this new approach to determine ΦCoupling Car S1-Chl directly from single TPE spectra and present first examples using known experimental spectra from Cars, Chl a, Chl b, LHC II, and PS 1.
Subject(s)
Carotenoids , Photosynthetic Reaction Center Complex Proteins , Chlorophyll , Energy Transfer , Light-Harvesting Protein Complexes , PhotonsABSTRACT
In the past decade, we developed various fluorescence-based methods for monitoring membrane fusion, membrane docking, distances between membranes, and membrane curvature. These tools were mainly developed using liposomes as model systems, which allows for the dissection of specific interactions mediated by, for example, fusion proteins. Here, we provide an overview of these methods, including two-photon fluorescence cross-correlation spectroscopy and intramembrane Förster energy transfer, with asymmetric labelling of inner and outer membrane leaflets and the calibrated use of transmembrane energy transfer to determine membrane distances below 10 nm. We discuss their application range and their limitations using examples from our work on protein-mediated vesicle docking and fusion.
Subject(s)
Membrane Fusion , Fluorescence , LiposomesABSTRACT
We present a comparison of the energy transfer between carotenoid dark states and chlorophylls for the minor complexes CP24 and CP29. To elucidate the potential involvement of certain carotenoid-chlorophyll coupling sites in fluorescence quenching of distinct complexes, varying carotenoid compositions and mutants lacking chlorophylls at specific binding sites were examined. Energy transfers between carotenoid dark states and chlorophylls were compared using the coupling parameter, [Formula: see text], which is calculated from the chlorophyll fluorescence observed after preferential carotenoid two-photon excitation. In CP24, artificial reconstitution with zeaxanthin leads to a significant reduction in the chlorophyll fluorescence quantum yield, [Formula: see text], and a considerable increase in [Formula: see text]. Similar effects of zeaxanthin were also observed in certain samples of CP29. In CP29, also the replacement of violaxanthin by the sole presence of lutein results in a significant quenching and increased [Formula: see text]. In contrast, the replacement of violaxanthin by lutein in CP24 is not significantly increasing [Formula: see text]. In general, these findings provide evidence that modification of the electronic coupling between carotenoid dark states and chlorophylls by changing carotenoids at distinct sites can significantly influence the quenching of these minor proteins, particularly when zeaxanthin or lutein is used. The absence of Chl612 in CP24 and of Chl612 or Chl603 in CP29 has a considerably smaller effect on [Formula: see text] and [Formula: see text] than the influence of some carotenoids reported above. However, in CP29 our results indicate slightly dequenching and decreased [Formula: see text] when these chlorophylls are absent. This might indicate that both, Chl612 and Chl603 are involved in carotenoid-dependent quenching in isolated CP29.
Subject(s)
Carotenoids/metabolism , Chlorophyll/metabolism , Darkness , Energy Transfer , Light-Harvesting Protein Complexes/metabolism , Binding Sites , Models, Molecular , Mutation/genetics , Spectrophotometry, UltravioletABSTRACT
Nonphotochemical quenching (NPQ) provides an essential photoprotection in plants, assuring safe dissipation of excess energy as heat under high light. Although excitation energy transfer (EET) between chlorophyll (Chl) and carotenoid (Car) molecules plays an important role in NPQ, detailed information on the EET quenching mechanism under in vivo conditions, including the triggering mechanism and activation dynamics, is very limited. Here, we observed EET between the Chl Q y state and the Car S1 state in high-light-exposed spinach thylakoid membranes. The kinetic and spectral analyses using transient absorption (TA) spectroscopy revealed that the Car S1 excited state absorption (ESA) signal after Chl excitation has a maximum absorption peak around 540 nm and a lifetime of â¼8 ps. Snapshot TA spectroscopy at multiple time delays allowed us to track the Car S1 ESA signal as the thylakoid membranes were exposed to various light conditions. The obtained snapshots indicate that maximum Car S1 ESA signal quickly rose and slightly dropped during the initial high-light exposure (<3 min) and then gradually increased with a time constant of â¼5 min after prolonged light exposure. This suggests the involvement of both rapidly activated and slowly activated mechanisms for EET quenching. 1,4-Dithiothreitol (DTT) and 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) chemical treatments further support that the Car S1 ESA signal (or the EET quenching mechanism) is primarily dependent on the accumulation of zeaxanthin and partially dependent on the reorganization of membrane proteins, perhaps due to the pH-sensing protein photosystem II subunit S.
ABSTRACT
Membrane fusion is an essential process in nature and is often accomplished by the specific interaction of SNARE proteins. SNARE model systems, in which SNARE domains are replaced by small artificial units, represent valuable tools to study membrane fusion in vitro. The synthesis and analysis is presented of SNARE model peptides that exhibit a recognition motif composed of two different types of peptide nucleic acid (PNA) sequences. This novel recognition unit is designed to mimic the SNARE zippering mechanism that initiates SNARE-mediated fusion. It contains N-(2-aminoethyl)glycine-PNA (aeg-PNA) and alanyl-PNA, which both recognize the respective complementary strand but differ in duplex topology and duplex formation kinetics. The duplex formation of PNA hybrid oligomers as well as the fusogenicity of the model peptides in lipid-mixing assays were characterized and the peptides were found to induce liposome fusion. As an unexpected discovery, peptides with a recognition unit containing only five aeg-PNA nucleo amino acids were sufficient and most efficient to induce liposome fusion.
Subject(s)
Liposomes/chemistry , Peptide Nucleic Acids/chemistry , Peptides/chemistry , SNARE Proteins/chemistry , Circular Dichroism , Liposomes/metabolism , Membrane Fusion , Models, Molecular , Peptide Nucleic Acids/metabolism , Peptides/metabolism , SNARE Proteins/metabolismABSTRACT
The photosystem II (PSII) subunit S (PsbS) plays a key role in nonphotochemical quenching, a photoprotective mechanism for dissipation of excess excitation energy in plants. The precise function of PsbS in nonphotochemical quenching is unknown. By reconstituting PsbS together with the major light-harvesting complex of PSII (LHC-II) and the xanthophyll zeaxanthin (Zea) into proteoliposomes, we have tested the individual contributions of PSII complexes and Zea to chlorophyll (Chl) fluorescence quenching in a membrane environment. We demonstrate that PsbS is stable in the absence of pigments in vitro. Significant Chl fluorescence quenching of reconstituted LHC-II was observed in the presence of PsbS and Zea, although neither Zea nor PsbS alone was sufficient to induce the same quenching. Coreconstitution with PsbS resulted in the formation of LHC-II/PsbS heterodimers, indicating their direct interaction in the lipid bilayer. Two-photon excitation measurements on liposomes containing LHC-II, PsbS, and Zea showed an increase of electronic interactions between carotenoid S1 and Chl states, Φ(Coupling)(CarS1-Chl), that correlated directly with Chl fluorescence quenching. These findings are in agreement with a carotenoid-dependent Chl fluorescence quenching by direct interactions of LHCs of PSII with PsbS monomers.
Subject(s)
Arabidopsis Proteins/metabolism , Chlorophyll/metabolism , Light-Harvesting Protein Complexes/metabolism , Photosystem II Protein Complex/metabolism , Recombinant Proteins/metabolism , Xanthophylls/metabolism , Arabidopsis , Blotting, Western , Chromatography, High Pressure Liquid , Circular Dichroism , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Liposomes/metabolism , Pisum sativum , Protein Folding , Spectrometry, Fluorescence , ZeaxanthinsABSTRACT
Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane.
Subject(s)
Membrane Fusion , SNARE Proteins/physiology , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/physiology , Animals , Cattle , Endocytosis , Fluorescence Polarization , Fluorescence Resonance Energy Transfer , ProteolipidsABSTRACT
It is known that aggregation of isolated light-harvesting complex II (LHCII) in solution results in high fluorescence quenching, reduced chlorophyll fluorescence lifetime, and increased electronic coupling of carotenoid (Car) S1 and chlorophyll (Chl) Qy states, as determined by two-photon studies. It has been suggested that this behavior of aggregated LHCII mimics aspects of non-photochemical quenching processes of higher plants and algae. However, several studies proposed that the minor photosystem II proteins CP24 and CP29 also play a significant role in regulation of photosynthesis. Therefore, we use a simple protocol that allows gradual aggregation also of CP24 and CP29. Similarly, as observed for LHCII, aggregation of CP24 and CP29 also leads to increasing fluorescence quenching and increasing electronic Car S1-Chl Qy coupling. Furthermore, a direct comparison of the three proteins revealed a significant higher electronic coupling in the two minor proteins already in the absence of any aggregation. These differences become even more prominent upon aggregation. A red-shift of the Qy absorption band known from LHCII aggregation was also observed for CP29 but not for CP24. We discuss possible implications of these results for the role of CP24 and CP29 as potential valves for excess excitation energy in the regulation of photosynthetic light harvesting.
Subject(s)
Carotenoids/metabolism , Chlorophyll Binding Proteins/metabolism , Light-Harvesting Protein Complexes/metabolism , Photosynthesis , Photosystem II Protein Complex/metabolism , Plants/metabolism , Carbon Isotopes/analysis , Carotenoids/chemistry , Chlorophyll/chemistry , Chlorophyll/metabolism , Chlorophyll Binding Proteins/chemistry , Chlorophyta/chemistry , Chlorophyta/metabolism , Fluorescence , Light-Harvesting Protein Complexes/chemistry , Photosystem II Protein Complex/chemistry , Plant Proteins/chemistry , Plant Proteins/metabolism , Plants/chemistry , Rhodophyta/chemistry , Rhodophyta/metabolismABSTRACT
In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca(2+) triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in cis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca(2+)-dependent and -independent cis-binding and trans-tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca(2+)-binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca(2+)-dependent cis-binding prevents trans-interactions if the cis-membrane contains 12-20% anionic phospholipids. Similarly, no trans-interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans-interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between cis- and trans-binding, which may play an important modulatory role in synaptic transmission.
Subject(s)
Presynaptic Terminals/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Synaptotagmin I/chemistry , Synaptotagmin I/metabolism , Animals , Binding Sites/physiology , Calcium/metabolism , Cell Membrane/metabolism , Liposomes/metabolism , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Rats , SNARE Proteins/metabolism , Spectrometry, Fluorescence , Synaptotagmin I/geneticsABSTRACT
Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1ß to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1ß with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1ß-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Histones/chemistry , Lysine/chemistry , Nucleosomes/chemistry , Biotinylation , Calorimetry/methods , Chromatin/chemistry , Chromatin/metabolism , Chromobox Protein Homolog 5 , DNA/chemistry , Epigenesis, Genetic , Histones/metabolism , Humans , Magnetic Resonance Spectroscopy/methods , Methylation , Nucleosomes/metabolism , Protein Binding , Protein Structure, Tertiary , Schizosaccharomyces/metabolismABSTRACT
Light-harvesting concentrators have a high potential to make highly efficient but precious energy converters, such as multijunction photovoltaics, more affordable for everyday applications. They collect sunlight, including diffusively scattered light, on large areas and redirect it to much smaller areas of the highly efficiency solar cells. Among the best current concepts are pools of randomly oriented light-collecting donor molecules that transfer all excitons to few aligned acceptors reemitting the light in the direction of the photovoltaics. So far, this system has only been realized for the 350-550 nm wavelength range, suitable for AlGaInP photovoltaics. This was achieved by using acceptor molecules that aligned during mechanical stretching of polymers together with donors, that stay random in that very same material and procedure. However, until recently, very little was known about the factors that are responsible for the alignability of molecules in stretched polymers and therefore it was difficult to find suitable donors and acceptors, as well as for other spectral ranges. Recently, a structural parameter was introduced with a high predictivity for the alignability of molecules that contain rigid band-like structures or linear aromatic π-systems. However, for light concentrators in more red spectral ranges, molecular systems often contain larger and extended, planar-like π-systems for which the previously reported parameter is not directly applicable. Here, we present a refined prediction parameter also suitable for larger plane-like structures. The new parameter depends on the number of in-plane atoms divided by out-of-plane atoms as determined by computational geometry optimization and additionally the planar aspect ratio for molecules that contain only in-plane atoms. With the help of this parameter, we found a new system that can efficiently collect and redirect light for the second 500-700 nm AlGaAs layer of current world-record multijunction photovoltaics. Similarly, as the previously reported system for the blue-green layer, it has also overall absorption and re-directioning quantum efficiencies close to 80-100%. Both layers, together, already cover about 75% of the energy in the solar spectrum.
ABSTRACT
An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP-based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization. Because of their simple protocols, they have become some of the most frequently used methods. However, standard fluorescent proteins present several drawbacks for sophisticated microscopy. With the HaloTag system, these drawbacks can be overcome, as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we have established the Split-HaloTag imaging assay in plants, which is based on the reconstitution of a functional HaloTag protein upon protein-protein interaction and the subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein-protein interaction at cellular structures: the anchoring of the molybdenum cofactor biosynthesis complex to filamentous actin. In addition, a specific interaction was visualized in a more distinctive manner with subdiffractional polarization microscopy, Airyscan, and structured illumination microscopy to provide examples of sophisticated imaging. Split-GFP and Split-HaloTag can complement one another, as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interactions using specific microscopy techniques, such as 3D imaging, single-molecule tracking, and super-resolution microscopy.
Subject(s)
Botany/instrumentation , Plants/metabolism , Protein Interaction Domains and MotifsABSTRACT
In higher plants, PsbS is known to play a key role in the regulation of photosynthetic light harvesting. However, the molecular mechanism and role of electronic carotenoid-chlorophyll (Chl) interactions for the downregulation of excess excitation (nonphotochemical energy quenching, NPQ) are still poorly understood. Here, we explored carotenoid â Chl energy transfer in isolated grana thylakoid membranes from mutants either deficient in or overexpressing PsbS. Since it was suggested that PsbS regulates the supramolecular protein network to control NPQ, we varied this network by diluting the grana protein densities. Our results indicate that different electronic quenching mechanisms are operative in grana thylakoids: a PsbS-dependent mechanism and a membrane protein density-dependent mechanism that is also operative in the absence of PsbS.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/metabolism , Carotenoids/metabolism , Chlorophyll/metabolism , Light-Harvesting Protein Complexes/genetics , Photosystem II Protein Complex/genetics , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Energy Transfer , Light-Harvesting Protein Complexes/metabolism , Mutation , Photosystem II Protein Complex/metabolism , Thylakoids/metabolism , Up-RegulationABSTRACT
Efficient sunlight harvesting and re-directioning onto small areas has great potential for more widespread use of precious high-performance photovoltaics but so far intrinsic solar concentrator loss mechanisms outweighed the benefits. Here we present an antenna concept allowing high light absorption without high reabsorption or escape-cone losses. An excess of randomly oriented pigments collects light from any direction and funnels the energy to individual acceptors all having identical orientations and emitting ~90% of photons into angles suitable for total internal reflection waveguiding to desired energy converters (funneling diffuse-light re-directioning, FunDiLight). This is achieved using distinct molecules that align efficiently within stretched polymers together with others staying randomly orientated. Emission quantum efficiencies can be >80% and single-foil reabsorption <0.5%. Efficient donor-pool energy funneling, dipole re-orientation, and ~1.5-2 nm nearest donor-acceptor transfer occurs within hundreds to ~20 ps. Single-molecule 3D-polarization experiments confirm nearly parallel emitters. Stacked pigment selection may allow coverage of the entire solar spectrum.
ABSTRACT
We present a direct comparison of two-photon spectra of various carotenoid-tetrapyrrole dyads and phthalocyanines (Pc) as well as chlorophylls (Chl) in the spectral range between 950 and 1360 nm, corresponding to one-photon spectra between 475 and 680 nm. For carotenoids (Car) with 8, 9, or 10 conjugated double bonds, the two-photon absorption cross section of states below the optical allowed carotenoid S2 is at least about 3-10 times higher than that of Pc or chlorophyll a and b at 550/1100 nm. A quantitative comparison of spectra from Pc with and without carotenoids of eight and nine conjugated double bonds confirms energy transfer from optically forbidden carotenoid states to Pc in these dyads. When considering that less than 100% efficient energy transfer reduces the two-photon contribution of the carotenoids in the spectra, the actual Car two-photon cross sections relative to Chl/Pc are even higher than a factor of 3-10. In addition, strong spectroscopic two-photon signatures at energies below the optical allowed carotenoid S2 state support the presence of additional optical forbidden carotenoid states such as S*, Sx, or, alternatively, contributions from higher vibronic or hot S1 states dominating two-photon spectra or energy transfer from the carotenoids. The onset of these states is shifted about 1500-3500 cm-1 to lower energies in comparison to the S2 states. Our data provides evidence that two-photon excitation of the carotenoid S*, Sx, or hot S1 states results in energy transfer to tetrapyrroles or chlorophylls similar to that observed with the Car S1 two-photon excitation.
Subject(s)
Carotenoids/chemistry , Chlorophyll/chemistry , Photons , Spectrophotometry , Tetrapyrroles/chemistry , Molecular StructureABSTRACT
The fusion of two opposing membranes is essential in biological functions such as fertilization, viral entry, membrane trafficking and synaptic transmission. Before the membrane bilayers are fully connected, at some stage a hemifusion intermediate-when the outer leaflets are merged but not the inner leaflets-is formed. However, the position of hemifusion in the energy landscape and the duration of it vary and have not been fully mapped out. To date, there has not been a way to differentiate lipid mixing of the two leaflets directly in a single experiment. Herein we demonstrate labeling of the outer and inner leaflets with different fluorophores, which can be distinguished by their fluorescence lifetimes. As a proof of concept, the asymmetrically labeled liposomes were used as acceptor liposomes in a novel one donor-two acceptor Förster resonance energy transfer (FRET) assay to monitor membrane fusion reactions mediated by the synaptic proteins soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) in microfluidic devices. Initial hemifusion was clearly indicated by the acceptor fluorescence lifetime originating solely from FRET acceptors on the outer leaflet (Oregon Green 488; τFl â¼ 4.8 ns). Progression to full fusion was then indicated by the significantly increasing lifetime contribution from acceptors on the inner leaflet (nitrobenzoxadiazole; τFl â¼ 6.7 ns). The new labeling strategy creates many possibilities in the design of bulk and single-molecule experiments.
Subject(s)
Fluorescence Resonance Energy Transfer , Lipid Bilayers/chemistry , Oxadiazoles/chemistry , Fluorescence , Liposomes/chemistryABSTRACT
Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1ß is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1ß bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1ß genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin.