Effect of phosphorylation on the thermal and light stability of the thylakoid membranes

Higher plant thylakoid membranes contain a protein kinase that phosphorylates certain threonine residues of light-harvesting complex II (LHCII), the main light-harvesting antenna complexes of photosystem II (PSII) and some other phosphoproteins (Allen, Biochim Biophys Acta 1098:275, 1992). While it has been established that phosphorylation induces a conformational change of LHCII and also brings about changes in the lateral organization of the thylakoid membrane, it is not clear how phosphorylation affects the dynamic architecture of the thylakoid membranes. In order to contribute to the elucidation of this complex question, we have investigated the effect of duroquinol-induced phosphorylation on the membrane ultrastructure and the thermal and light stability of the chiral macrodomains and of the trimeric organization of LHCII. As shown by small angle neutron scattering on thylakoid membranes, duroquinol treatment induced a moderate (~10%) increase in the repeat distance of stroma membranes, and phosphorylation caused an additional loss of the scattering intensity, which is probably associated with the partial unstacking of the granum membranes. Circular dichroism (CD) measurements also revealed only minor changes in the chiral macro-organization of the complexes and in the oligomerization state of LHCII. However, temperature dependences of characteristic CD bands showed that phosphorylation significantly decreased the thermal stability of the chiral macrodomains in phosphorylated compared to the non-phosphorylated samples (in leaves and isolated thylakoid membranes, from 48.3°C to 42.6°C and from 47.5°C to 44.3°C, respectively). As shown by non-denaturing PAGE of thylakoid membranes and CD spectroscopy on EDTA washed membranes, phosphorylation decreased by about 5°C, the trimer-to-monomer transition temperature of LHCII. It also enhanced the light-induced disassembly of the chiral macrodomains and the monomerization of the LHCII trimers at 25°C. These data strongly suggest that phosphorylation of the membranes considerably facilitates the heat- and light-inducible reorganizations in the thylakoid membranes and thus enhances the structural flexibility of the membrane architecture.     

Thermo-optically Induced Reorganizations in the Main Light Harvesting Antenna of Plants. II. Indications for the Role of LHCII-only Macrodomains in Thylakoids

We have investigated the circular dichroism spectral transients associated with the light-induced reversible reorganizations in chirally organized macrodomains of pea thylakoid membranes and loosely stacked lamellar aggregates of the main chlorophyll a/b light harvesting complexes (LHCII) isolated from the same membranes. These reorganizations have earlier been assigned to originate from a thermo-optic effect. According to the thermo-optic mechanism, fast local thermal transients due to dissipation of the excess excitation energy induce elementary structural changes in the close vicinity of the dissipation [Cseh et al. (2000) Biochemistry 39: 15250–15257]. Here we show that despite the markedly different CD spectra in the dark, the transient (light-minus-dark) CD spectra associated with the structural changes induced by high light in thylakoids and LHCII are virtually indistinguishable. This, together with other close similarities between the two systems, strongly suggests that the gross short-term, thermo-optically induced structural reorganizations in the membranes occur mainly, albeit probably not exclusively, in the LHCII-only domains [Boekema et al. (2000) J Mol Biol 301: 1123–1133]. Hence, LHCII-only domains might play an important role in light adaptation and photoprotection of plants.     

Thermo-optically Induced Reorganizations in the Main Light Harvesting Antenna of Plants. I. Non-Arrhenius Type of Temperature Dependence and Linear Light-intensity Dependencies

Thermo-optically induced structural reorganizations have earlier been identified in isolated LHCII, the main chlorophyll a/b light harvesting complexes of Photosystem II, and in granal thylakoid membranes [Cseh et al. (2000) Biochemistry 39: 15250–15257; Garab et al. (2002) Biochemistry 41: 15121–15129]. According to the thermo-optic mechanism, structural changes can be induced by fast, local thermal transients due to the dissipation of excess excitation energy. In this paper, we analyze the temperature and light-intensity dependencies of thermo-optically induced reversible and irreversible reorganizations in the chiral macrodomains of lamellar aggregates of isolated LHCII and of granal thylakoid membranes. We show that these structural changes exhibit non-Arrhenius type of temperature dependencies, which originate from the ‘combination’ of the ambient temperature and the local thermal transient. The experimental data can satisfactorily be simulated with the aid of a simple mathematical model based on the thermo-optic effect. The model also predicts, in good accordance with experimental data published earlier and presented in this paper, that the reorganizations depend linearly on the intensity of the excess light, a unique property that is probably important in light adaptation and photoprotection of plants.     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-beta,D-maltoside, n-octyl-beta,D-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

Light-induced trimer to monomer transition in the main light-harvesting antenna complex of plants: thermo-optic mechanism

The main chlorophyll a/b light-harvesting complex of photosystem II, LHCIIb, has earlier been shown to be capable of undergoing light-induced reversible structural changes and chlorophyll a fluorescence quenching in a way resembling those observed in granal thylakoids when exposed to excess light [Barzda, V., et al. (1996) Biochemistry 35, 8981-8985]. The nature and mechanism of this unexpected structural flexibility has not been elucidated. In this work, by using density gradient centrifugation and nondenaturing green gel electrophoresis, as well as absorbance and circular dichroic spectroscopy, we show that light induces a significant degree of monomerization, which is in contrast with the preferentially trimeric organization of the isolated complexes in the dark. Monomerization is accompanied by a reversible release of Mg ions, most likely from the outer loop of the complexes. These data, as well as the built-in thermal and light instability of the trimeric organization, are explained in terms of a simple theoretical model of thermo-optic mechanism, effect of fast thermal transients (local T-jumps) due to dissipated photon energies in the vicinity of the cation binding sites, which lead to thermally assisted elementary structural transitions. Disruption of trimers to monomers by excess light is not confined to isolated trimers and lamellar aggregates of LHCII but occurs in photosystem II-enriched grana membranes, intact thylakoid membranes, and whole plants. As indicated by differences in the quenching capability of trimers and monomers, the appearance of monomers could facilitate the nonphotochemical quenching of the singlet excited state of chlorophyll a. The light-induced formation of monomers may also be important in regulated proteolytic degradation of the complexes. Structural changes driven by thermo-optic mechanisms may therefore provide plants with a novel mechanism for regulation of light harvesting in excess light.     

Thermooptic effect in chloroplast thylakoid membranes. Thermal and light stability of pigment arrays with different levels of structural complexity

In chloroplast thylakoid membranes, chiral macrodomains, i.e., large arrays of pigment molecules with long-range chiral order, have earlier been shown to undergo light-induced reversible and irreversible structural changes; such reorganizations did not affect the short-range, excitonic pigment-pigment interactions. These structural changes and similar changes in lamellar aggregates of the main chlorophyll a/b light-harvesting complexes exhibited a linear dependence on the intensity of light that was not utilized in photosynthesis. It has been hypothesized that the light-induced rearrangements are driven by a thermooptic effect, i.e., thermal fluctuations due to the dissipation of excess excitation energies [Barzda, V., et al. (1996) Biochemistry 35, 8981-8985]. To test this hypothesis, we have utilized circular dichroism (CD) spectroscopy to investigate the structural stability of the chiral macrodomains and the constituent bulk pigment-protein complexes of granal thylakoid membranes against heat and prolonged, intense illumination. (i) In intact thylakoid membranes, the chiral macrodomains displayed high stability below 40 degrees C, but they were gradually disassembled between 50 and 60 degrees C; the thermal stability of the chiral macrodomains could be decreased substantially by suspending the membranes in reaction media that were hypotonic or had low ionic strength. (ii) The chiral macrodomains were also susceptible to high light: prolonged illumination with intense white light (25 min, 2500 microE m(-)(2) s(-)(1), 25 degrees C) induced similar, irreversible disassembly to that observed at high temperatures; in different preparations, lower thermal stability was coupled to lower light stability. (iii) The light stability depended significantly on the temperature: between about 5 and 15 degrees C, the macrodomains in the intact thylakoids were virtually not susceptible to high light; in contrast, the same preillumination at 35-40 degrees C almost completely destroyed the chiral macrodomains. (iv) As testified by the excitonic CD bands, the molecular organization of the pigment-protein complexes in all samples exhibited very high thermal stability between about 15 and 65 degrees C, and virtually total immunity against intense illumination. These data are fully consistent with the hypothesis of a thermooptic effect, and are interpreted within the frame of a simple model.     

Diurnal Fluctuations in the Content and Functional Properties of the Light Harvesting Chlorophyll a/b Complex in Thylakoid Membranes : Correlation with the Diurnal Rhythm of the mRNA Level

Diurnal fluctuations were observed in the content and some structural and functional properties of the light-harvesting chlorophyll (Chl) a/b pigment-protein complex of photosystem II (LHCII) in young developing wheat (Triticum aestivum) leaves grown under 16 hours light/8 hours dark illumination regime. The fluctuations could be correlated with the diurnal oscillation in the level of mRNA for LHCII. The most pronounced changes occurred in the basal segments of the leaves. They were weaker or hardly discernible in the middle and tip segments. As judged from the diurnal variations of the Chl-a/Chl-b molar ratio, the LHCII content of the thylakoid membranes peaked around 2 pm. This can be accounted for by the cumulative effect of the elevated level of mRNA in the morning and early afternoon. In the basal segment, the extent of the fluctuation in the LHCII content was approximately 25%, as determined from gel electrophoresis (“green gels”). The amplitude of the principal bands of the circular dichroism (CD) spectra of isolated chloroplasts paralleled the changes in the LHCII content. Our circular dichroism data suggest that the newly synthesized LHCII complexes are incorporated into the existing helically organized macrodomains of the pigment-protein complexes or themselves form such macrodomains in the thylakoid membranes. Chl-a fluorescence induction kinetics also showed diurnal variations especially in the basal segments of the leaves. This most likely indicates fluctuations in the ability of membranes to undergo “state transitions.” These observations suggest a physiological role of diurnal rhythm of mRNA for LHCII in young developing leaves.     

Study of pH and temperature-induced transitions in urate oxidase (Uox-EC1.7.3.3) by microcalorimetry (DSC), size exclusion chromatography (SEC) and enzymatic activity experiments

Purified recombinant urate oxidase (urate oxygen oxidoreductase EC 1.7.3.3. re-Uox) has been studied by means of differential scanning calorimetry (DSC) in correlation with enzymatic activity measurements and size exclusion chromatography. Differential scanning calorimetry curves versus pH show two endothermal effects in the pH range 6-10. The first endotherm reveals a maximum stability between pH 7.25 and pH 9.5 corresponding to a temperature of transition T(m1) of 49.0 degrees C and an enthalpy of transition of 326 kJ mol(-1). This value dramatically decreases below pH 7.25. The behavior of the second endotherm is more complex but the temperature of transition T(m2) is constant between pH 9 and 7.25 and a maximum for the corresponding enthalpy is obtained near pH 8 with DeltaH(2)=272 kJ mol(-1). An optimal pH of 8.0 for the stability of the enzymatic activity at elevated temperature was also found which was in good agreement with calorimetric results. Reversibility of the first endotherm is obtained from 20 to 51.5 degrees C. The calorimetric result is correlated to enzymatic activity, purity by size exclusion chromatography (SEC) and protein concentration measurements. In contrast, for the second endotherm, after heating up to 68.9 degrees C, no reversibility was found. Interaction with structural analogues of urate has been studied by DSC. 8-Azahyooxanthine has only a small effect and caffeine has no effect at all. With 8-azaxanthine, a rapid increase of the T(m1) function of the concentration is obtained. At high concentration T(m1) reached the T(m2) value which remained unaffected.     

Elevated temperature treatment induced alteration in thylakoid membrane organization and energy distribution between the two photosystems in Pisum sativum

Two-week-old pea (Pisum sativum var. Arkal) plants were subjected to elevated temperature (38 degrees C/42 degrees C) in dark for 14-15 h. The effect of heat treatment on light-induced phosphorylation of LHCII and LHCII migration in the thylakoid membranes were investigated. The heat treatment did cause a substantial (more than two fold) increase in the extent of LHCII phosphorylation as compared to the control. Upon separation of appressed and non-appressed thylakoid fractions by digitonin treatment, the heat-treated samples showed a decrease in LHCII-related polypeptides from the grana stack (appressed region) over the control. Further, a small increase in the intensity of these (LHCII-related) bands was detected in stromal thylakoid fraction (non-appressed membranes). This suggests an enhanced extent of migration of phosphorylated LHCII from appressed to non-appressed regions due to in vivo heat treatment of pea plants. We also isolated the LHCII from control and heat treated (42 degrees C) pea seedlings. Analysis of CD spectra revealed a 5-6 nm blue shift in the 638 nm negative peak in heat treated samples suggesting alteration in the organization of Chl b in the LHCII macro-aggregates. These results suggest that in vivo heat stress not only alters the extent of migration of LHCII to stromal region, but also affects the light harvesting mechanism by LHCII associated with the grana region.     

Protein dynamics and lipid affinity of monomeric,zeaxanthin-binding LHCII in thylakoid membranes

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from ¹³C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.     

Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin

The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.     

Pigment Interactions in Light-harvesting Complex II in Different Molecular Environments

Extraction of plant light-harvesting complex II (LHCII) from the native thylakoid membrane or from aggregates by the use of surfactants brings about significant changes in the excitonic circular dichroism (CD) spectrum and fluorescence quantum yield. To elucidate the cause of these changes, e.g. trimer-trimer contacts or surfactant-induced structural perturbations, we compared the CD spectra and fluorescence kinetics of LHCII aggregates, artificial and native LHCII-lipid membranes, and LHCII solubilized in different detergents or trapped in polymer gel. By this means we were able to identify CD spectral changes specific to LHCII-LHCII interactions, at (−)-437 and (+)-484 nm, and changes specific to the interaction with the detergent n-dodecyl-β-maltoside (β-DM) or membrane lipids, at (+)-447 and (−)-494 nm. The latter change is attributed to the conformational change of the LHCII-bound carotenoid neoxanthin, by analyzing the CD spectra of neoxanthin-deficient plant thylakoid membranes. The neoxanthin-specific band at (−)-494 nm was not pronounced in LHCII in detergent-free gels or solubilized in the α isomer of DM but was present when LHCII was reconstituted in membranes composed of phosphatidylcholine or plant thylakoid lipids, indicating that the conformation of neoxanthin is sensitive to the molecular environment. Neither the aggregation-specific CD bands, nor the surfactant-specific bands were positively associated with the onset of fluorescence quenching, which could be triggered without invoking such spectral changes. Significant quenching was not active in reconstituted LHCII proteoliposomes, whereas a high degree of energetic connectivity, depending on the lipid:protein ratio, in these membranes allows for efficient light harvesting.     

Identification of thylakoid membrane thermal transitions in Synechocystis sp. PCC6803 photosynthetic mutants

We used differential scanning calorimetry (DSC) as a technique capable of identifying photosynthetic complexes on the basis of their calorimetric transitions. Annotation of thermal transitions was carried out with thylakoid membranes isolated from various photosynthetic mutants of Synechocystis sp. PCC6803. The thylakoid membranes exhibited seven major DSC bands between 40 and 85°C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherms and by a subsequent annealing procedure. The successive annealing procedure proved to be more reliable technique than mathematical deconvolution in assigning thermal transitions. The main DSC band, around 47°C, resulting from the high enthalpy change that corresponds to non-interacting complex of PSII, was assigned using the PSI-less/apcE(-) mutant cells. Another band around 68-70°C relates to the denaturation of PSII surrounded by other proteins of the photosynthetic complexes in wild type and PSI-less/apcE(-) cells. A further major transition found at 82-84°C corresponds to the PSI core complex of wild type and PSII-deficient BE cells. Other transition bands between 50-67 and 65-75°C are believed to relate to ATP synthase and cytochrome b(6)f, respectively. These thermal transitions were obtained with thylakoids isolated from PSI(-)/PSII(-) mutant cells. Some minor bands determined at 59 and 83-84°C correspond to an unknown complex and NADH dehydrogenase, respectively. These annotations were done by PSI-less/apcE(-) and PSI(-)/PSII(-) mutants.     

Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane

Conversion of solar energy into chemical energy in plant chloroplasts concomitantly modifies the thylakoid architecture and hierarchical interactions between pigment–protein complexes. Here, the thylakoids were isolated from light‐acclimated Arabidopsis leaves and investigated with respect to the composition of the thylakoid protein complexes and their association into higher molecular mass complexes, the largest one comprising both photosystems (PSII and PSI) and light‐harvesting chlorophyll a/b‐binding complexes (LHCII). Because the majority of plant light‐harvesting capacity is accommodated in LHCII complexes, their structural interaction with photosystem core complexes is extremely important for efficient light harvesting. Specific differences in the strength of LHCII binding to PSII core complexes and the formation of PSII supercomplexes are well characterized. Yet, the role of loosely bound L‐LHCII that disconnects to a large extent during the isolation of thylakoid protein complexes remains elusive. Because L‐LHCII apparently has a flexible role in light harvesting and energy dissipation, depending on environmental conditions, its close interaction with photosystems is a prerequisite for successful light harvesting in vivo. Here, to reveal the labile and fragile light‐dependent protein interactions in the thylakoid network, isolated membranes were subjected to sequential solubilization using detergents with differential solubilization capacity and applying strict quality control. Optimized 3D‐lpBN‐lpBN‐sodium dodecyl sulfate–polyacrylamide gel electrophoresis system demonstrated that PSII–LHCII supercomplexes, together with PSI complexes, hierarchically form larger megacomplexes via interactions with L‐LHCII trimers. The polypeptide composition of LHCII trimers and the phosphorylation of Lhcb1 and Lhcb2 were examined to determine the light‐dependent supramolecular organization of the photosystems into megacomplexes.     

Functional domain size in aggregates of light-harvesting complex II and thylakoid membranes

The functional domain size for efficient excited singlet state quenching was studied in artificial aggregates of the main light-harvesting complex II (LHCIIb) from spinach and in native thylakoid membranes by picosecond time-resolved fluorescence spectroscopy and quantum yield measurements. The domain size was estimated from the efficiency of added exogenous singlet excitation quenchers-phenyl-p-benzoquinone (PPQ) and dinitrobenzene (DNB). The mean fluorescence lifetimes τ(av) were quantified for a range of quencher concentrations. Applying the Stern-Volmer formalism, apparent quenching rate constants k(q) were determined from the dependencies on quencher concentration of the ratio τ(0)(av)/τ(av), where τ(0)(av) is the average fluorescence lifetime of the sample without addition of an exogenous quencher. The functional domain size was gathered from the ratio k(q)'/k(q), i.e., the apparent quenching rate constants determined in aggregates (or membranes), k(q)', and in detergent-solubilised LHCII trimers, k(q), respectively. In LHCII macroaggregates, the resulting values for the domain size were 15-30 LHCII trimers. In native thylakoid membranes the domain size was equivalent to 12-24 LHCII trimers, corresponding to 500-1000 chlorophylls. Virtually the same results were obtained when membranes were suspended in buffers promoting either membrane stacking or destacking. These domain sizes are orders of magnitude smaller than the number of physically connected pigment-protein complexes. Therefore our results imply that the physical size of an antenna system beyond the numbers of a functional domain size has little or no effect on improving the light-harvesting efficiency.     

A calorimetric analysis of human plasma fibronectin: effects of heparin binding on domain structure

Fibronectin domain structure, as influenced by interaction with heparin, calcium, or chondroitin sulfate C, was analyzed by differential scanning calorimetry. A complex thermal denaturation transition was observed with a large sharp endotherm at 63 degrees C, a broad endotherm between 70 and 80 degrees C, and an exotherm at 80-90 degrees C. Analysis of the denaturation profiles revealed the existence of four thermal transitions, 59.1, 62.2, 67.3, and 74.3 degrees C, and an exotherm at 83.9 degrees C. The calorimetric enthalpies of the four endotherms are 1146 +/- 259, 866 +/- 175, 1010 +/- 361, and 676 +/- 200 kcal/mol, respectively. In all cases, the calorimetric to van't Hoff enthalpy ratio was greater than 1.0. Computer analysis of the primary structure of fibronectin revealed 29 +/- 8% homology among the type I homology units and 28 +/- 7% homology among type III homology units, suggesting that different structural domains could arise from the same homology type. This may explain why more thermal transitions are observed for fibronectin than there are homology types. Addition of heparin to fibronectin in varying molar ratios, i.e., 10:1 to 30:1, resulted in a larger calorimetric enthalpy for the first type of structural domain (Tm = 59.1 degrees C) of fibronectin. At higher heparin to fibronectin ratios (40:1 or 75:1), the enthalpy of this domain decreased, while the others remained unchanged. In the presence of 5 mM calcium chloride, fibronectin thermal denaturation occurred at lower temperatures and was associated with precipitation of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll-protein complexes in bean thylakoid membranes

Changes in chloroplast structure and rearrangement of chlorophyll-protein (CP) complexes were investigated in detached leaves of bean (Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 degrees C and subsequent 3-h photoactivation under white light (200 mumol photons m(-2) s(-1)) at 22 degrees C. Although, no change in chlorophyll (Chl) content and Chl a/b ratio in all samples was observed, overall fluorescence intensity of fluorescence emission and excitation spectra of thylakoid membranes isolated from dark-chilled leaves decreased to about 50%, and remained after photoactivation at 70% of that of the control sample. Concomitantly, the ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120 K increased 1.5-fold upon chilling, and was fully reversed after photoactivation. Moreover, chilling stress seems to induce a decrease of the relative contribution of LHCII fluorescence to the thylakoid emission spectra at 120 K, and an increase of that from LHCI and PSI, correlated with a decrease of stability of LHCI-PSI and LHCII trimers, shown by mild-denaturing electrophoresis. These effects were reversed to a large extent after photoactivation, with the exception of LHCII, which remained partly in the aggregated form. In view of these data, it is likely that dark-chilling stress induces partial disassembly of CP complexes, not completely restorable upon photoactivation. These data are further supported by confocal laser scanning fluorescence microscopy, which showed that regular grana arrangement observed in chloroplasts isolated from control leaves was destroyed by dark-chilling stress, and was partially reconstructed after photoactivation. In line with this, Chl a fluorescence spectra of leaf discs demonstrated that dark-chilling caused a decrease of the quantum yield PSII photochemistry (F(v)/F(m)) by almost 40% in 5 days. Complete restoration of the photochemical activity of PSII required 9 h post-chilling photoactivation, while only 3 h were needed to reconstruct thylakoid membrane organization and chloroplast structure. The latter demonstrated that the long-term dark-chilled bean leaves started to suffer from photoinhibition after transfer to moderate irradiance and temperature conditions, delaying the recovery of PSII photochemistry, independently of photo-induced reconstruction of PSII complexes.     

Anisotropic circular dichroism signatures of oriented thylakoid membranes and lamellar aggregates of LHCII

In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment–pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while “psi-type” CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.