Wojciech Grudzinski

Xanthophyll pigments in light-harvesting complex II in monomolecular layers: localisation, energy transfer and orientation

Monomolecular layers of the largest light-harvesting pigment-protein complex of Photosystem II (LHCII) were formed at the argon-water interface. The molecular area of the LHCII monomer in monomolecular layers determined from the isotherms of compression is found to be close to 14 nm2, which corresponds well to the molecular dimensions of the protein evaluated on the basis of crystallographic studies. Monolayers of LHCII were deposited on a glass support by means of the Langmuir-Blodgett technique and subjected to spectroscopic studies: electronic absorption spectrophotometry and spectrofluorometry. The fluorescence excitation spectra of chlorophyll a in monolayers of LHCII were analysed using gaussian deconvolution. Comparison of the absorption and fluorescence excitation spectra enabled calculation of the rate of excitation energy transfer in the system. Excitation energy was found to be transferred to chlorophyll a from chlorophyll b with 97% efficiency, from neoxanthin with 85%, from lutein with 62% and from violaxanthin with at least 54% efficiency. The analysis of the position of the 0-0 absorption band of the xanthophylls revealed that neoxanthin is located in the same protein environment as lutein but in a different environment than violaxanthin. The analysis of fluorescence excitation spectra of chlorophyll a in LHCII, recorded with the excitation light beam polarised in two orthogonal directions, enabled the determination of the mean orientation angle of the accessory xanthophyll pigments with respect to the plane of the sample. The mean orientation of lutein found in this study (approx. 51 degrees ) corresponds well to the crystallographic data. Neoxanthin was found to adopt a similar orientation to lutein. The transition dipole moment of violaxanthin was found to form a mean angle of 71 degrees with the axis spanning two polar regions of the protein, perpendicular to the plane of the monolayer, suggesting planar orientation of this pigment with respect to the plane of the thylakoid membrane. These experimentally determined xanthophyll orientations are discussed in terms of importance of peripheral xanthophyll pigments in supramolecular organisation of LHCII and the operation of the xanthophyll cycle within the thylakoid membrane.

Light-induced change of configuration of the LHCII-bound xanthophyll (tentatively assigned to violaxanthin): a resonance Raman study.

Raman scattering spectra of light-harvesting complex LHCII isolated from spinach were recorded with an argon laser, tuned to excite the most red-absorbing LHCII-bound xanthophylls (514.5 nm). The intensity of the nu(4) band (at ca. 950 cm-1) corresponding to the out-of-plane wagging modes of the C-H groups in the resonance Raman spectra of carotenoids appears to be inversely dependent on the probing laser power density. This observation can be interpreted in terms of excitation-induced change of configuration of the protein-bound xanthophyll owing to the fact that the intensity of this particular band is diagnostic of a chromophore twisting resulting from its binding to the protein environment. The comparison of the shape of the nu(4) band of a xanthophyll involved in the light-induced spectral changes with the shape of the nu(4) band of the xanthophylls present in LHCII, reported in the literature, lets us conclude that, most probably, violaxanthin is a pigment that undergoes light-driven changes of molecular configuration but also the involvement of lutein may not be excluded. Possible physical mechanisms responsible for the configuration changes and physiological importance of the effect observed are discussed.

Molecular Architecture of Plant Thylakoids under Physiological and Light Stress Conditions: A Study of Lipid–Light-Harvesting Complex II Model Membranes

The organization of plant thylakoid membranes under physiological and light stress conditions was analyzed in studies of model membranes formed with galactolipids and LHCII. The results show adaptation of an organization pattern of lipid-protein membranes to better fulfill two opposite physiological functions: harvesting of light quanta versus quenching of excess energy. In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.

Sphingomyelin-rich domains are sites of lysenin oligomerization: implications for raft studies.

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.

Toward Understanding of Toxic Side Effects of a Polyene Antibiotic Amphotericin B: Fluorescence Spectroscopy Reveals Widespread Formation of the Specific Supramolecular Structures of the Drug

Amphotericin B (AmB) is a lifesaving polyene antibiotic used widely to treat deep-seated mycoses. Both the pharmaceutical effectiveness as well as toxic side effects depend on molecular organization of the drug. In the present study, we analyzed steady-state fluorescence, fluorescence anisotropy spectra, fluorescence lifetimes, and fluorescence anisotropy decays of AmB in the systems believed to ensure monomeric organization of the drug and in model lipid membranes. The results of the analyses show that in all of the systems studied, the drug appears in, at least, two spectral forms, interpreted as monomeric and aggregated. Spectroscopic and fluorescence lifetime characteristics of both forms are provided. Interpretation of the fluorescence anisotropy spectra of AmB incorporated into liposomes formed with dipalmitoylphosphatidylcholine let us conclude that monomers of the drug are more tightly bound to the lipid membranes as compared to the aggregates and that AmB aggregates destabilize the membrane structure. Structural model analysis, compared to the analysis of spectral shifts, leads to the conclusion that basic constituents of AmB aggregated structure is a tetramer composed of two hydrogen-bond-stabilized dimers, each dimer formed by molecules twisted by ca. 170°. The tetramer itself can span lipid bilayers and can act as a transmembrane ion channel. Specific aggregate formation of AmB has been concluded as a universal and ubiquitous form of molecular organization of the drug. This process is discussed in terms of toxic side effects of AmB.

Supramolecular Organization of the Main Photosynthetic Antenna Complex LHCII: A Monomolecular Layer Study

The light-harvesting pigment-protein complex LHCII is a main antenna complex of the photosynthetic apparatus of plants, responsible for collecting light energy and also for photoprotection against overexcitation-induced damage. Realization of both functions depends on molecular organization of the complex. Monolayer technique has been applied to address the problem of supramolecular organization of LHCII. Analysis of the isotherms of compression of monomolecular films formed at the argon-water interface shows that LHCII appears in two phases: one characterized by the specific molecular area characteristic of trimeric and one of monomeric organization of LHCII. Monolayers of LHCII were deposited by means of the Langmuir-Blodgett technique to solid supports and examined by means of AFM, FTIR, fluorescence spectroscopy, and fluorescence lifetime imaging microscopy (FLIM). FTIR analysis shows that organization of the trimers of LHCII within a monolayer is associated with formation of intermolecular hydrogen bonds between neighboring polypeptides. The linear-dichroism FTIR analysis reveals that polypeptide fragments involved in intermolecular interactions are oriented at an angle of 67 degrees with respect to the normal axis to the plane of the layer. Fluorescence and fluorescence lifetime analysis reveal that the organization of LHCII within monolayers is associated with formation of the low-lying excitonic energy levels that can be potentially responsible for excess excitation quenching. FLIM and AFM reveal heterogeneous organization of LHCII monolayers, in particular, formation of ring-like structures. The potential of LHCII to form molecular structures characterized by pigment excitonic interactions is discussed in terms of regulation of the photosynthetic accessory function and photoprotection against overexcitation-induced damage.

Blue-light-controlled photoprotection in plants at the level of the photosynthetic antenna complex LHCII

Plants have developed several adaptive regulatory mechanisms, operating at all the organization levels, to optimize utilization of light energy and to protect themselves against over-excitation-related damage. We report activity of a previously unknown possible regulatory mechanism that operates at the molecular level of the major photosynthetic pigment-protein complexes of plants, LHCII. This mechanism is driven exclusively by blue light, operates in the trimeric but not in the monomeric complex, and results in singlet excitation quenching leading to thermal energy dissipation. The conclusions are based on single molecule fluorescence lifetime analysis, direct measurements of thermal energy dissipation by photo-thermal spectroscopy, and on fluorescence spectroscopy. Possible molecular mechanisms involved in the blue-light-induced photoprotective effect are discussed, including xanthophyll photo-isomerization and the thermo-optic effect.

The negative feedback molecular mechanism which regulates excitation level in the plant photosynthetic complex LHCII: Towards identification of the energy dissipative state

Overexcitation of the photosynthetic apparatus is potentially dangerous because it can cause oxidative damage. Photoprotection realized via the feedback de-excitation in the pigment-protein light-harvesting complex LHCII, embedded in the chloroplast lipid environment, was studied with use of the steady-state and time-resolved fluorescence spectroscopy techniques. Illumination of LHCII results in the pronounced singlet excitation quenching, demonstrated by decreased quantum yield of the chlorophyll a fluorescence and shortening of the fluorescence lifetimes. Analysis of the 77K chlorophyll a fluorescence emission spectra reveals that the light-driven excitation quenching in LHCII is associated with the intensity increase of the spectral band in the region of 700nm, relative to the principal band at 680nm. The average chlorophyll a fluorescence lifetime at 700nm changes drastically upon temperature decrease: from 1.04ns at 300K to 3.63ns at 77K. The results of the experiments lead us to conclude that: (i) the 700nm band is associated with the inter-trimer interactions which result in the formation of the chlorophyll low-energy states acting as energy traps and non-radiative dissipation centers; (ii) the Arrhenius analysis, supported by the results of the FTIR measurements, suggests that the photo-reaction can be associated with breaking of hydrogen bonds. Possible involvement of photo-isomerization of neoxanthin, reported previously (Biochim. Biophys. Acta 1807 (2011) 1237-1243) in generation of the low-energy traps in LHCII is discussed.

Is It Beneficial for the Major Photosynthetic Antenna Complex of Plants To Form Trimers

The process of primary electric charge separation in photosynthesis takes place in the reaction centers, but photosynthesis can operate efficiently and fluently due to the activity of several pigment-protein complexes called antenna, which absorb light quanta and transfer electronic excitations toward the reaction centers. LHCII is the major photosynthetic pigment-protein antenna complex of plants and appears in the trimeric form. Several recent reports point to trimeric organization of LHCII as a key factor responsible for the chloroplast architecture via stabilization of granal organization of the thylakoid membranes. In the present work, we address the question of whether such an organization could also directly influence the antenna properties of this pigment-protein complex. Chlorophyll fluorescence analysis reveals that excitation energy transfer in LHCII is substantially more efficient in trimers and dissipative energy losses are higher in monomers. It could be concluded that trimers are exceptionally well suited to perform the antenna function. Possibility of fine regulation of the photosynthetic antenna function via the LHCII trimer-monomer transition is also discussed, based on the fluorescence lifetime analysis in a single chloroplast.

Carotenoid binding to proteins: Modeling pigment transport to lipid membranes

Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes.