Energy transfer by chlorophyll a in detergent micelles

The process of energy transfer was studied in the chlorophyll a-containing detergent micelle, serving as a possible model of the photosynthetic unit. Chlorophyll a was added to aqueous solutions of the detergent Triton X-100 and incorporated into the micelles. The energy transfer process was studied by investigating the concentration depolarization of fluorescence of chlorophyll a. On the basis of the experimental depolarization curves as well as the value of the Förster parameter $\text{R}{}_0\text{= 56}\text{A}\text{?}$ calculated from the overlap of absorption and fluorescence spectra it was concluded that energy transfer between chlorophyll a molecules in this model follows the Förstertype mechanism of inductive resonance. Furthermore it was found that the local concentration of chlorophyll a in the micelles is higher by 1–3 orders of magnitude than its overall concentration in the solution and by choosing the appropriate ratio between the concentration of chlorophyll a and the detergent it is possible to reach the in vivo chlorophyll concentration of 0.1 M within the micelles. Thus the chlorophyll-detergent micelle model may be applied as a model of the separate package-type photosynthetic unit.     

Energy transfer by chlorophyll beta in detergent micelles.

The concentration-dependent depolarization, concentration-dependent quenching, absorption and fluorescence spectra in solutions of chlorophyll beta-containing detergent micelles with Triton X-100 were studied in a concentration range of c equal to 0.4 muM-0.6mM chlorophyll beta and cd equal to 0.4-7.0 mM Triton X-100. The concentration-dependent depolarization obeys F?rster's theory of depolarization of fluorescence with a transfer distance parameter R0 equal to 43 plus or minus 2 A. The concentration-dependent quenching is described by an empirical formula for the relative fluorescence yield n/n0 equal to 1/[1+(c/c1/2)-2] given by Kelly and Porter (Kelly A. R. and Porter, G. (1970) Proc. R. Soc. Lond. Ser. A. 315, 149-161). With increasing chlorophyll beta concentration the red absorption band at 650 nm is shifted toward a longer wavelength and its width increases by 10nm, the intensity of the long wave fluorescence band increases about 720 nm. The results analysed in terms of these findings lead to the conclusions that chlorophyll beta molecules are (a) locally concentrated in the micelles up to the concentration range of in vivo conditions, (b) partly in an aggregated state capable for fluorescence, (c) the chlorophyll beta yields chlorophyll beta homotransfer may be about 3-26% of the homotransfer chlorophyll alpha yields chlorophyll-alpha depending on the ratio of their concentrations.     

Lutein-chlorophyll-a energy transfer in detergent micelles

Absorption, fluorescence and fluorescence excitation spectra were determined for equimolar mixed micellar detergent solutions of lutein and chlorophyll-a in the concentration range from 9·10^?6 to 1.8·10^?4 M, with detergent (triton-X100) concentrations from 3·-10^?4 to 7·10^?3 M. In the range of detergent concentrations studied the pigments incorporated into the detergent micelles attained a high local concentration (0.1 to 0.01 M), reminiscent of pigment concentration within the chloroplast. A lutein → chlorophyll-a energy transfer with an efficiency of about 15% was found in these systems. In dilute (9·10^?6 M) pigment solution with concentrated (7·10^?3 M) detergent practically no transfer is observed. The extent of aggregation and the efficiency of transfer depend on the composition of the system. The aggregation of chlorophyll-a is partly inhibited by lutein molecules. It is shown that the energy transfer efficiency as function of distance follows anr ^?3 relationship,R ₀ being 22 å.     

Fluorescence resonance energy transfer analysis of lipopolysaccharide in detergent micelles.

Bacterial endotoxins or lipopolysaccharides (LPS), cell wall components of gram-negative bacteria, are involved in septic shock. LPS consists of a lipid A tail attached to core and O-antigen polysaccharides, but little is known about the supramolecular structure of LPS in blood. We have developed an approach to locate donor and acceptor probes in sulfobetaine palmitate detergent micelles using steady-state and time-resolved fluorescence resonance energy transfer. C18-fluorescein and several LPS species of varying molecular weight labeled with fluorescein isothiocyanate (FITC-LPS) were the donor probes. Acceptor probes were 1,1-dilinoleyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (Fast C18-Dil, Ro approximately 68 A), and octadecyl B rhodamine chloride (C18-Rhd, Ro approximately 58 A). With either acceptor, the transfer was of similar high efficiency when FITC-LPS Salmonella minnesota Re 595 (2,500 mol wt, lacking both core and O-antigen) or C18-fluorescein were the fluorescent donor probes. Thus, the donor FITC-LPS with short polysaccharide chain S. minnesota Re 595 and the control donor C18-fluorescein appear to be close to the micelle surface. The transfer efficiency decreased as the molecular weight of the LPS increased. Separation distances between the longest FITC-LPS, S. minnesota (20,000 mol wt, with a long O-antigen), and the micelle were estimated to be 1.5 Ro or more (approximately 100 A), consistent with an extended conformation for the longer O-antigen polysaccharide chain in the detergent.     

Structural study of rhodopsin in detergent micelles by small-angle neutron scattering

Monodisperse solutions of bovine rhodopsin monomers, devoid of lipid, associated with a linear polyoxyethylene alcohol detergent have been prepared. The composition and homogeneity of these complexes have been determined by hydrodynamic characterisation. Each rhodopsin molecule is associated with about 110 monomers of the detergent. These rhodopsin-detergent complexes have been studied by small-angle neutron scattering. Partial or total deuteration of the detergent, as well as variation of the ²H₂O/H₂O ratio in the solvent, were used to eliminate the detergent—solvent contrast at various protein—solvent contrasts. The size and shape of the detergent micelle and of the rhodopsin-detergent complexes were shown to be independent of solvent or detergent deuteration. Mixture of selectively deuterated detergent molecules allowed us to obtain an homogeneous scattering density for the detergent part of the micelles and therefore to eliminate totally its contribution to the scattering when it is contrast matched. Neutron scattering from rhodopsin alone was then measured even in highly deuterated solvents, with low incoherent background, as for a water-soluble protein. Supplementary neutron scattering measurements on rhodopsin-dodecyl dimethylamine oxide micelles confirmed essentially the results reported by Yeager (1975). Analysis of the neutron scattering data indicates that most of the hydrophobic residues of rhodopsin form a compact region which has zero hydration, this probably being the part which is embedded in the disc membrane, and that the unhydrated rhodopsin molecule is asymmetrically arranged with respect to the membrane. Comparison with the results of a small-angle X-ray scattering study (Sardet et al., 1976) implies that the peripheral regions on both sides of the membrane are highly hydrated. Several schematic models are discussed.     

Heterodimerization of BAK and MCL-1 activated by detergent micelles

BAK is a key protein mediating mitochondrial outer membrane permeabilization; however, its behavior in the membrane is poorly understood. Here, we characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment and study their interaction by in vitro pulldown experiments, size exclusion chromatography, titration calorimetry, and NMR spectroscopy. The nonionic detergent IGEPAL has little impact on the structure of MCL-1 but induces a conformational change in BAK, whereby its BH3 region is able to engage the hydrophobic groove of MCL-1. Although the zwitterionic detergent CHAPS induces only minor conformational changes in both proteins, it is still able to initiate heterodimerization. The complex of MCL-1 and BAK can be disrupted by a BID-BH3 peptide, which acts through binding to MCL-1, but a mutant peptide, BAK-BH3-L78A, with low affinity for MCL-1 failed to dissociate the complex. The mutation L78A in BAK prevented binding to MCL-1, thus demonstrating the essential role of the BH3 region of BAK in its regulation by MCL-1. Our results validate the current models for the activation of BAK and highlight the potential value of small molecule inhibitors that target MCL-1 directly.     

Tryptophan octyl ester in detergent micelles of dodecylmaltoside: fluorescence properties and quenching by brominated detergent analogs

The fluorescence properties of tryptophan octyl ester (TOE), a hydrophobic model of Trp in proteins, were investigated in various mixed micelles of dodecylmaltoside (DM) and 7,8-dibromododecyl beta-maltoside (BrDM) or 10,11-dibromoundecanoyl beta-maltoside (BrUM). This study focuses on the mechanism via which these brominated detergents quench the fluorescence of TOE in a micellar system. The experiments were performed at a pH at which TOE is uncharged and almost completely bound to detergent micelles. TOE binding was monitored by its enhanced fluorescence in pure DM micelles or its quenched fluorescence in pure BrUM or BrDM micelles. In DM/BrUM and DM/BrDM mixed micelles, the fluorescence intensity of TOE decreased, as a nonlinear function of the molar fraction of brominated detergent, to almost zero in pure brominated detergent. The indole moiety of TOE is therefore highly accessible to the bromine atoms located on the detergent alkyl chain because quenching by bromines occurs by direct contact with the fluorophore. TOE is simultaneously poorly accessible to iodide (I(-)), a water-soluble collisional quencher. TOE time-resolved fluorescence intensity decay is heterogeneous in pure DM micelles, with four lifetimes (from 0.2 to 4.4 ns) at the maximum emission wavelength. Such heterogeneity may arise from dipolar relaxation processes in a motionally restricted medium, as suggested by the time-dependent (nanoseconds) red shift (11 nm) of the TOE emission spectrum, and from the existence of various TOE conformations. Time-resolved quenching experiments for TOE in mixed micelles showed that the excited-state lifetime values decreased only slightly with increases in the proportion of BrDM or BrUM. In contrast, the relative amplitude of the component with the longest lifetime decreased significantly relative to that of the short-lived species. This is consistent with a mainly static mechanism for the quenching of TOE by brominated detergents. Molecular modeling of TOE (in vacuum and in water) suggested that the indole ring was stabilized by folding back upon the octyl chain, forming a hairpin conformation. Within micelles, the presence of such folded conformations, making it possible for the entire molecule to be located in the hydrophobic part of the micelle, is consistent with the results of fluorescence quenching experiments. TOE rotational correlation time values, in the nanosecond range, were consistent with a hindered rotation of the indole moiety and a rotation of the complete TOE molecule in the pure DM or mixed detergent micelles. These results, obtained with a simple micellar model system, provide a basis for the interpretation of fluorescence quenching by brominated detergents in more complex systems such as protein- or peptide-detergent complexes.     

Energy transfer dynamics in a red-shifted violaxanthin-chlorophyll a light-harvesting complex

Photosynthetic eukaryotes whose cells harbor plastids originating from secondary endosymbiosis of a red alga include species of major ecological and economic importance. Since utilization of solar energy relies on the efficient light-harvesting, one of the critical factors for the success of the red lineage in a range of environments is to be found in the adaptability of the light-harvesting machinery, formed by the proteins of the light-harvesting complex (LHC) family. A number of species are known to employ mainly a unique class of LHC containing red-shifted chlorophyll a (Chl a) forms absorbing above 690?nm. This appears to be an adaptation to shaded habitats. Here we present a detailed investigation of excitation energy flow in the red-shifted light-harvesting antenna of eustigmatophyte Trachydiscus minutus using time-resolved fluorescence and ultrafast transient absorption measurements. The main carotenoid in the complex is violaxanthin, hence this LHC is labeled the red-violaxanthin-Chl a protein, rVCP. Both the carotenoid-to-Chl a energy transfer and excitation dynamics within the Chl a manifold were studied and compared to the related antenna complex, VCP, that lacks the red-Chl a. Two spectrally defined carotenoid pools were identified in the red antenna, contributing to energy transfer to Chl a, mostly via S₂ and hot S₁ states. Also, Chl a triplet quenching by carotenoids is documented. Two separate pools of red-shifted Chl a were resolved, one is likely formed by excitonically coupled Chl a molecules. The structural implications of these observations are discussed.     

Structure and thermal stability of monomeric bacteriorhodopsin in mixed phospholipid/detergent micelles

Thermal unfolding experiments on bacteriorhodopsin in mixed phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 +/- 13 A in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysozyme activity in the presence of nonionic detergent micelles

The effect of a nonionic surfactant, polyoxyethylenesorbitan monolaurate (Tween 20), on the hen egg-white lysozyme catalyzed lysis of a dried cell suspension of Micrococcus lysodeikticus is analysed. A rate enhancement of up to 70% is observed in the presence of surfactant at concentrations above the critical micelle concentration. This activity increase may be explained by postulating the existence of a micelle-enzyme complex in which enzyme molecules are bound to micelles with preferential orientation of their active sites. The reaction is found to be second order with respect to substrate. A mechanism is postulated in which a substrate particle is assumed to be an energy-furnishing collision partner to the enzyme-substrate complex. This mechanism correlated data over a wide range of enzyme and substrate concentrations. Data from kinetic, ultrafiltration, ultraviolet, and fluorescence studies provide convincing evidence for the existence of a micelle-lysozyme complex. The results suggest that it is possible that immobilized enzymes mat in general be more reactive than corresponding free enzymes.     

Energy transfer from photosystem II to photosystem I in Porphyridium cruentum

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of Porphyridium cruentum which had been frozen to ?196 °C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at ?196 °C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.     

Studies on Triton X-100 detergent micelles.

Triton X-100 micelle formation at 25 degrees C was studied by use of sedimentation equilibrium and fluorescence spectroscopic techniques. The apparent molecular weight of the major Triton X-100 micelle was found to be 81250, indicating a micelle number of 125. A micelle number of 121 was obtained with fluorescence titration experiments, which showed one molecule of 1-anilino-8-naphthalene sulfonate binding per micelle with an apparent association constant of 0.9 x 10(5) M. The fluorescent titration experiments also indicated the presence of another TX-100 binding species of variable size.     

Association of myelin basic protein with detergent micelles

Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being $\text{3.58 ± 0.12}\text{and}\text{2.30 ± 0.15}$ for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively, $\text{1.34 ± 0.10}$ for deoxycholate (at 0.12 ionic strength) and $\text{4.0 ± 0.5}$ for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strength in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and detergent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single polypeptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules.     

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P_Zn), the accessory Zn-BChl a (B_Zn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P_Zn^*, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B_Zn^* to P_Zn, and the latter, the electron transfer from P_Zn to H. The angle between the transition dipoles of B_Zn and P_Zn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P_Zn.     

Reaction center preparations of Rhodopseudomonas spheroides: Energy transfer and structure

1.

1. A procedure for the preparation of a reaction center fraction from wild type Rhodopseudomonas spheroides is described. The process involves two subsequent detergent treatments. The particles were purified down to a protein weight of 120000 daltons. They contain little cytochrome and 1.2 moles of ubiquinone per mole of P870. The negative absorption change in the light minus dark difference spectrum is not inconsistent with the assumption that 1 mole of ubiquinone is reduced per mole of photooxidized P870.     

Association of myelin basic protein with detergent micelles.

Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being 3.58 +/- 0.12 and 2.30 +/- 0.15 for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively 1.34 +/- 0.10 for deoxycholate (at 0.12 ionic strength) and 4.0 +/- 0.5 for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strenght in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and deterent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single poly-peptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules.     

NMR study of the tetrameric KcsA potassium channel in detergent micelles

Nuclear magnetic resonance (NMR) studies of large membrane-associated proteins are limited by the difficulties in preparation of stable protein-detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68-kDa homotetrameric KcsA, a thermostable N-terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C-terminal cytoplasmic domain, absent in the original structure, contains a 14-residue helix that could participate in tetramerization by forming an intersubunit four-helix bundle. A quantitative estimate of cross- relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS-KcsA mixed micelles form an oblate spheroid with approximately 180 SDS molecules per channel. K(+) ions bind to the micelle-solubilized channel with a K(D) of 3 +/- 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH-induced changes in chemical shift along the "outer" transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH-gating of the potassium channel.     

Antimalarial drugs and heme in detergent micelles: An NMR study

Proton nuclear magnetic resonance relaxation times were measured for the protons of micelles formed by the detergents sodium dodecyl sulfate, dodecyltrimethyl ammonium bromide, and polyethylene glycol sorbitan monolaureate in the presence of ferriprotoporphyrin IX and the antimalarial drugs chloroquine, 7-chloro-4-quinolyl 4-N,N-diethylaminobutyl sulfide, and primaquine. Diffusion coefficients were extracted from pulsed gradient NMR experiments to evaluate the degree of association of these drugs with the detergent micelles. Results indicate that at low or neutral pH when the quinolyl N is protonated, chloroquine does not associate with neutral or cationic detergent micelles. For this reason, chloroquine’s interaction with heme perturbs the partitioning of heme between the aqueous medium and detergent micelles.     

Towards structural investigations on isotope labelled native bacteriorhodopsin in detergent micelles by solution-state NMR spectroscopy

¹H NMR signals of the retinal moiety in detergent-solubilizedbacteriorhodopsin are assigned, enabling the interpretation of NOEs within thechromophore. To achieve this, a number of differently labelled samples wereprepared to test the applicability of the various assignment and distancemeasurement strategies. In measurements with and without light,¹H and ¹³C chemical shifts of the retinal in thenative protein were partially assigned for both the dark- and thelight-adapted states. Additionally, samples with residue-specific¹H amino acids and/or retinal in an otherwise deuterated proteinwere prepared to measure the distances between either two kinds of amino acidsor between individual amino acids and the retinal moiety. With the observationof NOE within the bound retinal and between retinal and its neighbouring aminoacids, an important step towards the elucidation of distance constraints inthe binding pocket of the proton pump is made.