Aggregation of chlorophylls in monolayers. Part IV. The reorganisation of chlorophyll a in multilayer array

The nature of the interaction between the chlorophyll a molecules in multilayer arrays obtained by the Langmuir-Blodgett technique is examined by electronic and infrared spectroscopies. Following the deposition of the multilayers, we observed a blue shift with time in the electronic spectra. This effect is monitored by infrared spectroscopy. The intensity of the coordinated ketone band is decreased while the intensity of the free ketone band is increased. These modifications are explained by the reorganization of the chlorophyll a molecules from an organized to a less organized one. The influence of H2O, D2O and SO2 vapors on the chlorophyll a multilayers give some informations on the role of water molecules in the aggregation of chlorophyll a in this ordered system. From these observations, a model is proposed for the multilayer arrangement implying two molecules of water per molecule of chlorophyll a.     

Aggregation of chlorophylls in monolayers. V. The effect of water on chlorophyll a and chlorophyll b in mono and multilayer arrays

The nature of the interactions between water molecules and monolayers and multilayers of chlorophyll a (Chl a), and monolayers and multilayers of Chl b. obtained by the Langmuir-Blodgett technique, is examined by infrared spectroscopy. Following deposition of the monolayer or multilayer of Chl a or Chl b onto a plate, repetitive scans showed some modifications in the infrared spectra which are interpreted as a reorganization of the molecules as some water molecules leave the array. Drying the sample further modifies the spectra, which indicates the departure of more tenacious water molecules. Putting the sample in a moist atmosphere does not restore the original spectrum. This is an indication of a nonreversibie reorganization in the chlorophyll array. The spectra of the monolayer of chlorophyll are much more complicated than those of the multilayer, owing to the nonintegrating effect of the monolayer, which reveals the perturbing effect of the different dielectric milieux on each functional group. On the basis of the analysis of the spectra and the information gathered from the surface pressure isotherms, a model is proposed for the monolayer arrangement at the air/water interface which implies two set of dimers of water per molecule of chlorophyll. One pair of dimers constitutes the water of the first kind and is composed of vapor-like dimers. This kind of water is situated between the porphyrin planes of chlorophyll molecules and is easily removed from the monolayer. The second pair of dimers is composed of water of the second and third kinds situated between the Mg atom of the chlorophyll molecules and the water of the subphase. The second kind of water is closest to the Mg atom and is the most difficult one to remove. The third kind of water is closest to the surface and its mobility is intermediate between that of water of the first kind and that of water of the second kind. Comparing the infrared spectra of a freshly prepared monolayers of Chl a with the resonance Raman spectra of intact chloroplasts (M. Lutz, Biochim. Biophys. Acta 460 (1977) 408), we notice great similarities. This is an indication that the model we propose for the monolayer of Chl a could play an important role in the chloroplast.     

Aggregation of gramicidin A in phospholipid Langmuir-Blodgett monolayers

The aggregation of Gramicidin A (gA) in dipalmitoylphosphatidylcoline (DPPC) monolayers is investigated by both thermodynamic and structural methods. Compression isotherm analysis and atomic force microscopy (AFM) observations are performed. Our experimental results indicate that gA aggregation does occur in DPPC monolayers even at very low gA concentration (about 8 x 10(-4) mol%). At the low gA concentration limit, the aggregation process seems to be mainly horizontal (i.e., side-by-side, into the monolayer plane), following a fractal pattern growth producing the formation of typical, flat (0.5 nm height) "doughnut" structures, with a diameter of approximately 150 nm. These structures appear to be composed of smaller subunits (about 70 nm diameter) showing the same doughnut structure. At a molar fraction of approximately 3.8 mol%, the big doughnuts start to disaggregate and only small doughnuts appear. Above a gA concentration of approximately 4.4 mol%, all doughnuts (large and small) disappear, and the morphology assumes the appearance of a patchwork of two distinct phases: one that, being very flat, can be associated with a gA-free or gA-poor DPPC phase, and a second one, characterized by a more corrugated surface, associated with a gA-rich DPPC phase. At gA concentration of approximately 5 mol%, a percolation transition in the gA-rich DPPC phase occurs. Thermodynamic data indicate that the maximum of miscibility between gA and DPPC molecules occurs at approximately 28 mol%, suggesting that gA could aggregate in hexamers that are, on average, bound to 16 DPPC molecules. At the same concentration, AFM images show a network of small gA aggregation units of a size compatible with gA hexamers.     

Aggregation of puroindoline in phospholipid monolayers spread at the air-liquid interface

Puroindolines, cationic and cystine-rich low molecular weight lipid binding proteins from wheat seeds, display unique foaming properties and antimicrobial activity. To unravel the mechanism involved in these properties, the interaction of puroindoline-a (PIN-a) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers was studied by coupling Langmuir-Blodgett and imaging techniques. Compression isotherms of PIN-a/phospholipid monolayers and adsorption of PIN-a to lipid monolayers showed that the protein interacted strongly with phospholipids, especially with the anionic DPPG. The electrostatic contribution led to the formation of a highly stable lipoprotein monolayer. Confocal laser scanning microscopy and atomic force microscopy showed that PIN-a was mainly inserted in the liquid-expanded phase of the DPPC, where it formed an aggregated protein network and induced the fusion of liquid-condensed domains. For DPPG, the protein partitioned in both the liquid-expanded and liquid-condensed phases, where it was aggregated. The extent of protein aggregation was related both to the physical state of phospholipids, i.e., condensed or expanded, and to the electrostatic interactions between lipids and PIN-a. Aggregation of PIN-a at air-liquid and lipid interfaces could account for the biological and technological properties of this wheat lipid binding protein.     

Lability of chlorophylls in solvent

Journal of Applied Phycology - DMSO (dimethyl sulfoxide, (CH3)2SO) is an alternative solvent for the spectroscopic assay of chlorophylls (Chl) but has mainly been used on Chl a and b organisms, but...     

Excitonic coupling of chlorophylls in the plant light-harvesting complex LHC-II.

Manifestation and extent of excitonic interactions in the red Chl-absorption region (Q(y) band) of trimeric LHC-II were investigated using two complementary nonlinear laser-spectroscopic techniques. Nonlinear absorption of 120-fs pulses indicates an increased absorption cross section in the red wing of the Q(y) band as compared to monomeric Chl a in organic solution. Additionally, the dependence of a nonlinear polarization response on the pump-field intensity was investigated. This approach reveals that one emitting spectral form, characterized by a 2.3(+/-0.8)-fold larger dipole strength than monomeric Chl a, dominates the fluorescence spectrum of LHC-II. Considering available structural and spectroscopic data, these results can be consistently explained assuming the existence of an excitonically coupled dimer located at Chl-bindings sites a2 and b2 (referring to the original notation of W. Nühlbrandt, D.N. Wang, and Y. Fujiyoshi, Nature, 1994, 367:614-621), which must not necessarily correspond to Chls a and b). This fluorescent dimer, terminating the excitation energy-transfer chain of the LHC-II monomeric subunit, is discussed with respect to its relevance for intra- and inter-antenna excitation energy transfer.     

Aggregation of isolated myofibrils stimulated by their contraction. II. Aggregation mechanism

Aggregation of contracted myofibrils was studied as a function of experimental conditions at myofibril contraction. The aggregation rate increased at higher concentrations of suspensions and at increased ionic strength of the medium to achieve the maximum at 0.1 M KCL in the last case. The aggregate sizes grow with an increase of ionic strength and concentration of MgATP and reduce with addition of F-actin. Aggregation of myofibrils develops only in the case of their complete or significant contraction. It was suggested that aggregation is stimulated by dehydration of myofibril at contraction.     

Lateral diffusion of cholesterol in monolayers.

The surface diffusion coefficient of cholesterol in cholesterol monolayers has been measured as a function of cholesterol surface concentration. Two different radiochemical methods, one integral and the other differential, were developed which gave comparable results. In the integral method two cholesterol monolayers, one of which is radioactive, are isolated on inert hydrophilic supports and then brought into contact. After some time the supports are separated and the radioactivity of the supports is measured. The differential method is an autoradiographic experiment. Two cholesterol monolayers, one of which is radioactive, are separated by means of a thin barrier. Upon removal of the barrier and at later times, an autoradiographic plate is brought to within a fraction of a mm from the aqueous surface and exposed. The plates are developed and analysed. The data show that the cholesterol surface diffusion coefficient in the dilute monolayers is approximately 10(-6)cm2/s and is nearly independent of surface concentration up to a concentration corresponding to an area of 40 A2/molecule. As the monolayer becomes compressed beyond this surface concentration, the diffusion coefficient decreases ubruptly with the deeply decreasing surface tension to about 10(-7) cm2/s, when a fully condensed surface layer of 38 A2/molecule is reached. This diffusion coefficient is of the same order of magnitude as the diffusion coefficients measured in lipid bilayers and in membranes.     

Photo-stable chlorophylls conjugated with montmorillonite

Summary Chlorophyll a was adsorbed to freeze-dried montmorillonite, bentonite, in benzene to form chlorophyll-bentonite conjugate. The adsorption proceeds into two steps; monolayer and multilayer adsorption. The absorption maximum in red region of chlorophyll a shifts to longer wavelength from 660 nm to 678 nm by increasing the amount of chlorophyll a conjugated with bentonite. Chlorophylls in the conjugated state was found to be photo-stable, although chlorophylls free from bentonite took place the marked bleaching by light-illumination.     

Minor but key chlorophylls in Photosystem II

A ‘metal-free’ chlorophyll (Chl) a, pheophytin (Phe) a, functions as the primary electron acceptor in PS II. On the basis of Phe a/PS II = 2, Phe a content is postulated as an index for estimation of the stoichiometry of pigments and photosystems. We found Phe a in a Chl d-dominant cyanobacterium Acaryochloris marina, whereas Phe d was absent. The minimum Chl a:Phe a ratio was 2:2, indicating that the primary electron donor is Chl a, accessory is Chl d, and the primary electron acceptor is Phe a in PS II of A. marina. Chl d was artificially formed by the treatment of Chl a with papain in aqueous organic solvents. Further, we will raise a key question on the mechanisms of water oxidation in PS II.     

Aggregation of tyrocidine in aqueous solutions.

The formation of aggregates of tyrocidine B at 4°C and 20°C in aqueous solutions was studied by means of light scattering and fluorescent techniques. The apparent weight molecular weight of tyrocidine B aggregates was found to be 36,000 at 4°C and 28,800 at 20°C. Fluorescence titration experiments with dansyl-chloride resulted in an aggregational number of 31 (4°) and 28 (20°) indicating that one molecule of dye is bound per monomer of molecular weight 1,200. From a Scatchard plot apparent association constants of 1.22 × 10⁵ M (4°) and 0.95 × 10⁵ M (20°) were calculated. From the angular dependence of scattered intensity the radii of gyration were determined to be 60 Å and 58 Å, respectively.     

Aggregation of deoxyhemoglobin subunits.

The formation of deoxyhemoglobin was examined by measuring the heme spectral change that accompanies the aggregation of isolated alpha and beta chains. At low hemeconcentrations (less than 10(-5) M), tetramer formation can be described by two consecutive, second order reactions representing the aggregation of monomers followed by the association of alphabeta dimers. At neutral pH, the rates of monomer and dimer aggregation are roughly the same, approximately 5 X 10(5) M(-1) X(-1) at 20 degrees. Raising or lowering the pH results in a uniform decrease of both aggregation rates due presumably to repulsion of positively charged subunits at acid pH and repulsion of negatively charged subunits at alkaline pH. Addition of p-hydroxymercuribenzoate to alpha chains lowers the rate of monomer aggregation whereas addition of mercurials to the beta subunits appears to lower both the rate of monomer and the rate of dimer aggregation. At high heme concentrations (greater than 10(-5) M) or in the presence of organic phosphates, the rate of chain aggregation becomes limited, in part, by the slow dissociation of beta chain tetramers. In the case of inositol hexaphosphate, the rate of hemoglobin formation exhibits a bell-shaped dependence on phosphate concentration. When intermediate concentrations of inositol hexaphosphate (approximately 10(-4 M) are preincubated with beta subunits, a slow first order time course is observed and exhibits a half-time of about 8 min. As more inositol hexaphosphate is added, the chain aggregation reaction begins to occur more rapidly. Eventually at about 10(-2) M inositol hexaphospate, the time course becomes almost identical to that observed in the absence of phosphates. The increase in the velocity of the chain aggregation reaction at high phosphate concentrations suggests strongly that inositol hexaphosphate binds to beta monomers and, if added in sufficiently large amounts, promotes beta4 dissociation. A quantitative analysis of these results showed that the affinity of beta monomers for inositol hexaphosphate is the same as that of alphabeta dimers. Only when tetramers are formed, either alpha2beta2 or beta4, is a marked increase in affinity for inositol hexaphosphate observed.     

Involvement of uncoupled antenna chlorophylls in photoinhibition in thylakoids

Evidence is presented, by means of both fluorescence and action spectroscopy, that a small, spectroscopically heterogeneous population of both Chl a and Chl b molecules is present in isolated spinach thylakoids and is active in photoinhibition. The broadness of the action spectrum suggests that degraded or incompletely assembled pigment-protein complexes may be involved.     

Selective photobleaching of PSI-related chlorophylls in heat-stressed pea chloroplasts

Measurements of electron transport activity point to the occurrence of major changes in the organisation of the photosynthetic apparatus of heat-stressed chloroplasts. One of the consequences of these changes is shown to be a greatly increased susceptibility of chlorophyll to photobleaching. Despite the fact that the threshold temperature for this photobleaching coincides closely with that for the inhibition of PSII activity, the bleached components were found to be specifically associated with PSI. This increased susceptibility of PSI pigments to photobleaching is shown to be a direct consequence of an interruption of the flow of reductants from PSII to PSI that would normally protect PSI from photooxidation.Abbreviations PSI photosystem I - PSII photosystem II - chl a chlorophyll a - chl b chlorophyll b - LHCP chlorophyll a/b light-harvesting protein - CP₁ P700-chlorophyll a protein - DCMU 3-(34 dichlorophenyl)-11-dimethylurea - DCPIP dichlorophenolindophenol - Fecy potassium ferricyanide - MV methyl viologen Biochemistry Department, King's College (KQC), University of London