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.     

Rapid reversible formation of a metastable subgel phase in saturated diacylphosphatidylcholines.

Formation of well ordered lamellar subgel (SGII) phase in aqueous dispersions of L-dipalmitoylphosphatidylcholine upon cooling from the lamellar gel phase, without low-temperature equilibration, is observed in real time using synchrotron x-ray diffraction. It has the same lamellar repeat period as the gel phase from which it was formed but differs in its wide-angle diffraction pattern. The SGII phase forms at about 7 degrees C upon cooling at 2 degrees C/min. In temperature jump experiments at 1 degree C/s from 50 to -5 degrees C, the relaxation time of the lamellar gel-SGII transition is found to be approximately 15 s. The conversion between the lamellar gel and SGII phase is cooperative and rapidly reversible. Upon heating, it coincides in temperature with an endothermic event with a calorimetric enthalpy of 0.35 kcal/mol, the so-called sub-subtransition. Similar sub-subtransitions are also observed calorimetrically at temperatures approximately 10 degrees C below the subtransition, without low-temperature storage, in aqueous dispersions of L-dimyristoylphosphatidylcholine and L-distearoylphosphatidylcholine, but not in racemic DL-dipalmitoylphosphatidylcholine. The formation of the equilibrium lamellar crystalline Lc phase appears to take place only from within the SGII phase.     

Phycobilisome integrity and functionality in lipid unsaturation and xanthophyll mutants in Synechocystis

Photosynthesis Research - The major light-harvesting system in cyanobacteria, the phycobilisome, is an essential component of the photosynthetic apparatus that regulates the utilization of the...     

Structural rearrangements in chloroplast thylakoid membranes revealed by differential scanning calorimetry and circular dichroism spectroscopy. Thermo-optic effect

The thermo-optic mechanism in thylakoid membranes was earlier identified by measuring the thermal and light stabilities of pigment arrays with different levels of structural complexity [Cseh, Z., et al. (2000) Biochemistry 39, 15250-15257]. (According to the thermo-optic mechanism, fast local thermal transients, arising from the dissipation of excess, photosynthetically not used, excitation energy, induce elementary structural changes due to the "built-in" thermal instabilities of the given structural units.) The same mechanism was found to be responsible for the light-induced trimer-to-monomer transition in LHCII, the main chlorophyll a/b light-harvesting antenna of photosystem II (PSII) [Garab, G., et al. (2002) Biochemistry 41, 15121-15129]. In this paper, differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy on thylakoid membranes of barley and pea are used to correlate the thermo-optically inducible structural changes with well-discernible calorimetric transitions. The thylakoid membranes exhibited six major DSC bands, with maxima between about 43 and 87 degrees C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherm and by a successive annealing procedure; these yielded similar thermodynamic parameters, transition temperature and calorimetric enthalpy. A systematic comparison of the DSC and CD data on samples with different levels of complexity revealed that the heat-induced disassembly of chirally organized macrodomains contributes profoundly to the first endothermic event, a weak and broad DSC band between 43 and 48 degrees C. Similarly to the main macrodomain-associated CD signals, this low enthalpy band could be diminished by prolonged photoinhibitory preillumination, the extent of which depended on the temperature of preillumination. By means of nondenaturing, "green" gel electrophoresis and CD fingerprinting, it is shown that the second main endotherm, around 60 degrees C, originates to a large extent from the monomerization of LHCII trimers. The main DSC band, around 70 degrees C, which exhibits the highest enthalpy change, and another band around 75-77 degrees C relate to the dismantling of LHCII and other pigment-protein complexes, which under physiologically relevant conditions cannot be induced by light. The currently available data suggest the following sequence of events of thermo-optically inducible changes: (i) unstacking of membranes, followed by (ii) lateral disassembly of the chiral macrodomains and (iii) monomerization of LHCII trimers. We propose that thermo-optical structural reorganizations provide a structural flexibility, which is proportional to the intensity of the excess excitation, while for their localized nature, the structural stability of the system can be retained.     

Structural integrity of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 phycobilisomes evaluated by means of differential scanning calorimetry

Phycobilisomes (PBSs) are supramolecular pigment–protein complexes that serve as light-harvesting antennae in cyanobacteria. They are built up by phycobiliproteins assembled into allophycocyanin core cylinders (ensuring the physical interaction with the photosystems) and phycocyanin rods (protruding from the cores and having light-harvesting function), the whole PBSs structure being maintained by linker proteins. PBSs play major role in light-harvesting optimization in cyanobacteria; therefore, the characterization of their structural integrity in intact cells is of great importance. The present study utilizes differential scanning calorimetry and spectroscopy techniques to explore for the first time, the thermodynamic stability of PBSs in intact Synechocystis sp. PCC 6803 cells and to probe its alteration as a result of mutations or under different growth conditions. As a first step, we characterize the thermodynamic behavior of intact and dismantled PBSs isolated from wild-type cells (having fully assembled PBSs) and from CK mutant cells (that lack phycocyanin rods and contain only allophycocyanin cores), and identified the thermal transitions of phycocyanin and allophycocyanin units in vitro. Next, we demonstrate that in intact cells PBSs exhibit sharp, high amplitude thermal transition at about 63 °C that strongly depends on the structural integrity of the PBSs supercomplex. Our findings implicate that calorimetry could offer a valuable approach for the assessment of the influence of variety of factors affecting the stability and structural organization of phycobilisomes in intact cyanobacterial cells.     

C-terminal functional unit of Rapana thomasiana (marine snail, gastropod) hemocyanin isoform RtH1: isolation and characterization

Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin (Hc) isoforms termed RtH1 and RtH2. Both subunit types are built up of eight functional units (FUs). The C-terminal functional unit (RtH1-h) of the Rapana Hc subunit 1 has been isolated by limited trypsinolysis of the subunit polypeptide chain. The oxy- and apo-forms of the unit are characterized by fluorescence spectroscopy. Upon excitation of RtH1-h at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein globule determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the indole emission of the oxy-RtH1-h. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-RtH1-h. The thermal stability of the apo-RtH1-h is characterized fluorimetrically by the "melting" temperature T(m) (65 degrees C) and by the transition temperature T(m) (83 degrees C) obtained by differential scanning calorimetry for oxy-RtH1-h. The results confirm the role of the copper-dioxygen complex for the stabilization of the Hc structure in solution.     

Differential scanning calorimetry of the irreversible denaturation of Rapana thomasiana (marine snail, Gastropod) hemocyanin

The thermal denaturation of the hemocyanin from gastropod Rapana thomasiana (RtH) at neutral pH was studied by means of differential scanning calorimetry (DSC). The denaturation was completely irreversible as judged by the absence of any endotherm on rescanning of previously scanned samples. Two transitions, with apparent transition temperatures (T(m)) at 83 and 90 degrees C, were detected by DSC using buffer 20 mM MOPS, containing 0.1 M NaCl, 5 mM CaCl(2) and 5 mM MgCl(2), pH 7.2. Both T(m) were dependent on the scanning rate, suggesting that the thermal denaturation of RtH is a kinetically controlled process. The activation energy (E(A)) of 597+/-20 kJ mol(-1) was determined for the main transition (at 83 degrees C). E(A) for the second transition was 615+/-25 kJ mol(-1). The T(m) and Delta H(cal) values for the thermal denaturation of RtH were found to be independent of the protein concentration, signifying that the dissociation of the protein into monomers does not take place before the rate-determining state of the process of thermal unfolding.     

Low pH Modulates the Macroorganization and Thermal Stability of PSII Supercomplexes in Grana Membranes

Protonation of the lumen-exposed residues of some photosynthetic complexes in the grana membranes occurs under conditions of high light intensity and triggers a major photoprotection mechanism known as energy dependent nonphotochemical quenching. We have studied the role of protonation in the structural reorganization and thermal stability of isolated grana membranes. The macroorganization of granal membrane fragments in protonated and partly deprotonated state has been mapped by means of atomic force microscopy. The protonation of the photosynthetic complexes has been found to induce large-scale structural remodeling of grana membranes—formation of extensive domains of the major light-harvesting complex of photosystem II and clustering of trimmed photosystem II supercomplexes, thinning of the membrane, and reduction of its size. These events are accompanied by pronounced thermal destabilization of the photosynthetic complexes, as evidenced by circular dichroism spectroscopy and differential scanning calorimetry. Our data reveal a detailed nanoscopic picture of the initial steps of nonphotochemical quenching.