Lipid rafts reconstituted in model membranes

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.     

Pigment and protein composition of reconstituted light-harvesting complexes and effects of some protein modifications

The structure and heterogeneity of LHC II were studied by in vitro reconstitution of apoproteins with pigments (Plumley and Schmidt 1987, Proc Natl Acad Sci 84: 146–150). Reconstituted CP 2 complexes purified by LDS-PAGE were subsequently characterized and shown to have spectroscopic properties and pigment-protein compositions and stoichiometries similar to those of authentic complexes. Heterologous reconstitutions utilizing pigments and light-harvesting proteins from spinach, pea and Chlamydomonas reinhardtii reveal no evidence of specialized binding sites for the unique C. reinhardtii xanthophyll loroxanthin: lutein and loroxanthin are interchangeable for in vitro reconstitution. Proteins modified by the presence of a transit peptide, phosphorylation, or proteolytic removal of the NH₂-terminus could be reconstituted. Evidence suggests that post-translational modification are not responsible for the presence of six electrophoretic variants of C. reinhardtii CP 2. Reconstitution is blocked by iodoacetamide pre-treatment of the apoproteins suggesting a role for cysteine in pigment ligation and/or proper folding of the pigment-protein complex. Finally, no effect of divalent cations on pigment reassembly could be detected.Abbreviations cab chlorophyll a/b-binding protein genes - Chl chlorophyll - CP2 light-harvesting chlorophyll A+b-protein complex fractionated by mildly denaturing LDS-PAGE from Photosystem II in thylakoids - CP 43 and CP 47 chlorophyll a-antenna complexes fractionated from Photosystem II in thylakoids by mildly denaturing LDS-PAGE at 4°C - IgG gamma immunoglobulin - LDS lithium dodecyl sulfate - LDS-PAGE lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C - LHC I and LHC II thylakoid light-harvesting chlorophyll a+b-protein holocomplexes associated with Photosystems I and II, respectively - PS II Photosystem II - TX100 Triton X-100 - TX100-derived LHC light-harvesting complexes enriched in LHC II following fractionation of thylakoids by TX100     

A comparative spectroscopic and kinetic study of photoexcitations in detergent-isolated and membrane-embedded LH2 light-harvesting complexes

Integral membrane proteins constitute more than third of the total number of proteins present in organisms. Solubilization with mild detergents is a common technique to study the structure, dynamics, and catalytic activity of these proteins in purified form. However beneficial the use of detergents may be for protein extraction, the membrane proteins are often denatured by detergent solubilization as a result of native lipid membrane interactions having been modified. Versatile investigations of the properties of membrane-embedded and detergent-isolated proteins are, therefore, required to evaluate the consequences of the solubilization procedure. Herein, the spectroscopic and kinetic fingerprints have been established that distinguish excitons in individual detergent-solubilized LH2 light-harvesting pigment-protein complexes from them in the membrane-embedded complexes of purple photosynthetic bacteria Rhodobacter sphaeroides. A wide arsenal of spectroscopic techniques in visible optical range that include conventional broadband absorption-fluorescence, fluorescence anisotropy excitation, spectrally selective hole burning and fluorescence line-narrowing, and transient absorption-fluorescence have been applied over broad temperature range between physiological and liquid He temperatures. Significant changes in energetics and dynamics of the antenna excitons upon self-assembly of the proteins into intracytoplasmic membranes are observed, analyzed, and discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Single-molecule visualization of environment-sensitive fluorophores inserted into cell membranes by staphylococcal gamma-hemolysin

Single-molecule imaging of the entrance of a protein into the hydrophobic environment of a cell membrane was investigated. The pre-stem of LukF, one of the two components of the pore-forming toxin staphylococcal gamma-hemolysin, was specifically labeled with 6-bromoacetyl-2-dimethylaminonaphthalene (Badan), an environment-sensitive fluorophore. Incubation of this derivative with erythrocyte ghost membranes resulted in a pronounced increase in fluorescence indicating insertion of Badan into the hydrophobic interior of the lipid bilayers. However, the increase in fluorescence was completely dependent on the interaction of Badan-labeled LukF with the gamma-hemolysin second component. Individual spots of Badan fluorescence on erythrocyte membranes were visualized that were associated with single pores. Analyses of the intensities of these fluorescent spots and their photobleaching independently showed that a single pore contained 3-4 LukF molecules. Thus, environment-sensitive fluorophore signals can be used to study the insertion of specific protein domains into cell membranes at the single-molecule lever, and the use of this approach in the present study revealed that a single gamma-hemolysin pore opening contains at least three LukF molecules.     

Single-molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation

Single-molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post-translational modifications. Here, we present a new method integrating single-molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28-mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which regulates let-7 microRNA biogenesis. Our real-time analysis of the uridylation by the TUT4 immunoprecipitates suggests that Lin28 functions as a processivity factor of TUT4. Our new technique, SIMPlex (single-molecule approach to immunoprecipitated protein complexes), provides a universal tool to analyse complex proteins at the single-molecule level.     

Single-molecule investigation of the interactions between reconstituted planar lipid membranes and an analogue of the HP(2-20) antimicrobial peptide

In this study, we employed electrophysiology experiments carried out at the single-molecule level to study the mechanism of action of the HPA3 peptide, an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Amplitude analysis of currents fluctuations induced by HPA3 peptide at various potentials in zwitterionic lipid membranes reveal the existence of reproducible conductive states in the stochastic behavior of such events, which directly supports the existence of transmembrane pores induced the peptide. From our data recorded both at the single-pore and macroscopic levels, we propose that the HPA3 pore formation is electrophoretically facilitated by trans-negative transmembrane potentials, and HPA3 peptides translocate into the trans monolayers after forming the pores. We present evidence according to which the decrease in the membrane dipole potential of a reconstituted lipid membranes leads to an augmentation of the membrane activity of HPA3 peptides, and propose that a lower electric dipole field of the interfacial region of the membrane caused by phloretin facilitates the surface-bound HPA3 peptides to break free from one leaflet of the membrane, insert into the membrane and contribute to pore formation spanning the entire thickness of the membrane.     

The low-energy forms of photosystem I light-harvesting complexes: spectroscopic properties and pigment-pigment interaction characteristics

In this work the spectroscopic properties of the special low-energy absorption bands of the outer antenna complexes of higher plant Photosystem I have been investigated by means of low-temperature absorption, fluorescence, and fluorescence line-narrowing experiments. It was found that the red-most absorption bands of Lhca3, Lhca4, and Lhca1-4 peak, respectively, at 704, 708, and 709 nm and are responsible for 725-, 733-, and 732-nm fluorescence emission bands. These bands are more red shifted compared to "normal" chlorophyll a (Chl a) bands present in light-harvesting complexes. The low-energy forms are characterized by a very large bandwidth (400-450 cm(-1)), which is the result of both large homogeneous and inhomogeneous broadening. The observed optical reorganization energy is untypical for Chl a and resembles more that of BChl a antenna systems. The large broadening and the changes in optical reorganization energy are explained by a mixing of an Lhca excitonic state with a charge transfer state. Such a charge transfer state can be stabilized by the polar residues around Chl 1025. It is shown that the optical reorganization energy is changing through the inhomogeneous distribution of the red-most absorption band, with the pigments contributing to the red part of the distribution showing higher values. A second red emission form in Lhca4 was detected at 705 nm and originates from a broad absorption band peaking at 690 nm. This fluorescence emission is present also in the Lhca4-N-47H mutant, which lacks the 733-nm emission band.     

The spirilloxanthine-pathway carotenoid incorporation into light-harvesting complexes of Ectothiorhodospira haloalkaliphila in vitro

Carotenoidless light-harvesting complexes (DPA-complexes) LH1-RC and LH2 were isolated from the purple sulfur bacterium Ectothiorhodospira haloalkaliphila in which carotenoid biosynthesis was suppressed with diphenylamine (DPA). Carotenoids of the spirilloxanthine series, which were isolated from the same bacterium, were incorporated into the DPA-complexes in vitro with an efficiency of 95–100%. The comparison of characteristics of the complexes with the incorporated carotenoids and the control complexes showed that the LH2 complexes with the incorporated carotenoids restored their absorption spectra, circular dichroism signals, and energy transfer from carotenoids to bacteriochlorophyll, which indicates that carotenoids were correctly incorporated into the structure of this complex.     

Thermal stability studies of photosystem II complexes reconstituted into phosphatidylcholine liposomes

Light-harvesting II complexes (LHCII) and photosystem II core complexes (PSIICC) were isolated from spinach (Spinacia oleracea L.) and reconstituted into phosphatidylcholine liposomes and, under heat stress, PSIICC-LHCII proteoliposomes were found to exhibit significantly higher oxygen evolution activity than PSIICC proteoliposomes lacking LHCII. In the presence of LHCII, the temperature of a 10-min heat stress that caused semi-inactivation of oxygen-evolving activity in these liposomes increased from 34 to ～37°C and the total inactivation temperature increased from ～50 to ～60°C. Moreover, with heat stress, decreases in the absorbance and fluorescence spectra of PSIICC-LHCII proteoliposomes were smaller than in LHCII-lacking PSIICC proteoliposomes. These results demonstrated that reconstitution of PSII into liposomes with LHCII increased the antenna size and light harvesting cross-section of PSII and thus, under heat stress, enhanced PSII photochemical activity and thermal stability.     

Single-molecule spectroscopic determination of lac repressor-DNA loop conformation

The Escherichia coli lactose repressor protein (LacI) provides a classic model for understanding protein-induced DNA looping. LacI has a C-terminal four-helix bundle tetramerization domain that may act as a flexible hinge. In previous work, several DNA constructs, each containing two lac operators bracketing a sequence-induced bend, were designed to stabilize different possible looping geometries. The resulting hyperstable LacI-DNA loops exist as both a compact "closed" form with a V-shaped repressor and also a more "open" form with an extended hinge. The "9C14" construct was of particular interest because footprinting, electrophoretic mobility shift, and ring closure experiments suggested that it forms both geometries. Previous fluorescence resonance energy transfer (FRET) measurements gave an efficiency of energy transfer (ET) of 70%, confirming the existence of a closed form. These measurements could not determine whether open form or intermediate geometries are populated or the timescale of interconversion. We have now applied single-molecule FRET to Cy3, Cy5 double-labeled LacI-DNA loops diffusing freely in solution. By using multiple excitation wavelengths and by carefully examining the behavior of the zero-ET peak during titration with LacI, we show that the LacI-9C14 loop exists exclusively in a single closed form exhibiting essentially 100% ET.     

Spectral dynamics of individual bacterial light-harvesting complexes: alternative disorder model

The bacterial (Rhodopseudomonas acidophila) photosynthetic peripheral light-harvesting complex of type 2 (LH2) exhibits rich fluorescence spectral dynamics at room temperature. The fluorescence spectrum of individual LH2 shifts either to the blue or to the red during the experimental observation time of a few minutes. These spectral changes are often reversible and occur between levels of a distinctly different peak wavelength. Furthermore, they are accompanied by a change of the spectral line shape. To interpret the dynamics of spectral changes, an energetic disorder model associated with easily explainable structural changes of the protein is proposed. This model assumes that each pigment in the tightly coupled ring of bacteriochlorophylls can be in two states of electronic transition energy due to the protein-pigment interaction. The transition between these structural, and hence spectroscopic, states occurs through the thermally induced conformational potential energy barrier crossing. Although simplified, the model allows us to reproduce the bulk fluorescence spectrum, the distribution of the single-molecule spectral peak wavelength and its changes, and the statistics of the duration of the spectral states. It also provides an intuitively clear picture of possible protein dynamics in LH2. At the same time, it requires additional sophistication since it essentially does not reproduce the red occurrences of single LH2 spectra.     

Molecular assembly of Zn porphyrin complexes using synthetic light-harvesting model polypeptides

Synthetic single α-helix hydrophobic polypeptides, which have similar amino acid sequences to the hydrophobic core in the native light-harvesting 1-β polypeptide from Rhodobacter sphaeroides, formed Zn porphyrin complexes on a gold electrode, as well as in n-octyl-β-glucoside micelles: this process is dependent on the structure of the pigments and the polypeptides. Interestingly, an enhanced photoelectric current was observed when Zn mesoporphyrin monomer complexed with the synthetic light-harvesting model polypeptide in an α-helical configuration was assembled with a defined orientation onto the electrode. Analog of these light-harvesting model complexes are also useful in providing insights into the effect of polypeptide structure on the formation of light-harvesting complexes on and off electrodes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Single-molecule electron tunneling spectroscopy of the higher plant light-harvesting complex LHC II

Electronic spectroscopy of a single biological molecule is demonstrated with approximately 4 A spatial resolution. The light-harvesting complex II (LHC II), in the ground and photo-excited states, was studied using scanning tunneling microscopy and spectroscopy of intact Photosystem II complexes. Analysis of the spectra indicates that the main mechanisms of tunneling between the STM tip and the surface involve delocalized electronic states of the LHC II and local vibronic states associated with C=C, C=O, C-H, N-H, and O-H groups near the LHC II surface. Conduction within the bulk LHC II is then due to ohmic and hopping conduction as well as tunneling between amino acid residues. Light activation of LHC II occurs via a photoconductive rather than a photovoltaic mechanism. There is a dramatic light-induced increase in the electronic density of states indicating a light-induced enhancement of energy and electron delocalization which is important for the efficient and rapid transfer of excitation energy from LHC II to the Photosystem II reaction center.     

Fluorescence spectroscopic studies of pressure effects on Na+,K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures

To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na+,K+-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated. To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order. Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers. Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.     

Erythrocyte D-glucose transport activity in reconstituted model membranes of different lipid composition

The stereospecific influx of D-glucose into liposomes formed on sonication of different glyco- and phospholipids with transport proteins from human erythrocyte ghosts solubilized with Triton x-100 was measured as an index of their total D-glucose transport activity. Specific D-glucose transport increased when acidic phospho- and glycolipids (especially sulfatide) were added to the phosphatidylcholine bilayers of the model membranes while cholesterol strongly inhibited the process. The modulation of D-glucose transport activity and its possible correlation with the lipid composition and the chemico-physical state of the erythrocytes is discussed.     

Reconstitution of light-harvesting complexes and photosystem II cores into galactolipid and phospholipid liposomes

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 to that described for LHC-PC liposomes. Reconstitution of PSII cores into DG vesicles also produced proteoliposomes with randomly dispersed particles (approximately 7.5 +/- 1.6 nm). In contrast, PSII-PC mixtures formed convoluted networks of tubular membranes that exhibited very few fracture faces. Most of the protein particles (approximately 7.0 +/- 1.5 nm) were seen trapped between, rather than embedded in, the membranes. The interaction between the zwitterionic head group of the phosphatidyl choline and the negatively charged PSII core may be responsible for the unusual membrane structures observed.     

Effect of ethanol on tracheal potassium channels reconstituted into bilayer lipid membranes.

We examined the effect of ethanol on single potassium channels derived from plasma membranes of bovine tracheal smooth muscles. The observed potassium channels had a conductance of 296 +/- 31 pS (mean +/- S.D.) in symmetrical 250 mmol/l KCl solutions, and exhibited a voltage- and Ca2+-dependence similar to BKCa channels. Ethanol at 50, 100 and 200 mM concentrations increased the probability of open potassium channels to 112 +/- 5, 127 +/- 7 and 121 +/- 13% (mean +/- S.E.M.), respectively. It is suggested that increased activity of the BKCa channels by ethanol hyperpolarizes the plasma membrane and thus may contribute to relaxation of tracheal smooth muscle.