Color of illumination during growth affects LHCII chiral macroaggregates in pea plant leaves.

To determine whether the color of illumination under which plants are grown, affects the structure of photosynthetic antennae, pea plants were grown under either blue-enriched, red-enriched, or white light. Carotenoid content of isolated chloroplasts was found to be insensitive to the color of illumination during growth, while chlorophyll a/b ratio in chloroplasts isolated from young illuminated leaves showed susceptibility to color. Color of illumination affects the LHCII chiral macroaggregates in intact leaves and isolated chloroplasts, providing light-induced alteration of the handedness of the LHCII chiral macroaggregate, as measured with circular dichroism and circularly polarized luminescence. The susceptibility of handedness to current illumination (red light excitation of chlorophyll fluorescence) is dependent on the color under which the plants were grown, and was maximal for the red-enriched illumination. We propose the existence of a long-term (growth period) color memory, which influences the susceptibility of the handedness of LHCII chiral macroaggregates to current light.

Left- and right-handed LHC II macroaggregates revealed by circularly polarized chlorophyll luminescence.

Circularly polarized chlorophyll luminescence (CPL) may serve as a measure of chiral macroaggregates of the light-harvesting chlorophyll-protein complexes (LHC II) in both isolated chloroplasts and intact leaves (Gussakovsky et al (2000) Photosynth Res 65: 83-92). In the present work, we applied the CPL approach to study the effect of fast (1-2 min) thermal impacts on LHC II macroaggregates. The results revealed unexpected temperature-response kinetics, composed of initial bell-shaped changes in the CPL signal, followed by degradation down to a steady state (equilibrium). The bell-shape effect was dependent upon illumination, and vanished in the dark. A mathematical analysis of the temperature-response kinetics uniquely indicated that LHC II chiral macroaggregates may persist in both left- and right-handed forms. These forms differ in their response to high temperatures. Both forms are more thermostable in leaves than in isolated chloroplasts. The cooperative degradation of LHC II macroaggregates, which is induced by the thermal impact, is irreversible. It is therefore suggested that the native LHC II macroaggregates are stable, stationary, non-equilibrium, spatially heterogeneous (dissipative) structures. The dissipative properties probably allow the interconversion between left- and right-handed forms under perturbation by certain factors. Illumination probably serves as one such perturbation factor, initiating the interconversion of dark-adapted, left-handed to light-dependent, right-handed LHC II macroaggregates. The chiral heterogeneity of the LHC II macroaggregates is a newly revealed aspect which needs to be taken into consideration in future circular dichroism or CPL studies.

Large-scale preparation of biologically active mouse and rat leptins and their L39A/D40A/F41A muteins which act as potent antagonists.

Expression plasmids encoding mouse and rat leptins and their L39A/D40A/F41A muteins were prepared. The proteins were expressed in Escherichia coli, refolded and purified to homogeneity, yielding electrophoretically pure, over 98% monomeric protein. Circular dichroism (CD) analysis revealed that the mutations hardly affect the leptins' secondary structure, and they were similar to previously reported CD spectra for human leptin. Both mouse and rat leptins were biologically active in promoting proliferation in BAF/3 cells stably transfected with the long form of human leptin receptor. The mutations did not change the binding properties to BAF/3 cells as compared, respectively, to non-mutated mouse, rat or human leptins, or their ability to form 1:1 complexes with the leptin-binding domain of chicken leptin receptor. In contrast, their biological activity, tested in a BAF/3 proliferation assay, was abolished and both became potent antagonists. As the LDF (amino acids 39-41) sequence is preserved in all known leptins, the present results substantiate the hypothesis that this sequence plays a pivotal role in leptins' site III and that interaction of leptin with its receptors resembles the corresponding interactions of interleukin-6 and granulocyte colony-stimulating factor their receptors.

Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists.

Interaction of leptin with its receptors resembles that of interleukin-6 and granulocyte colony-stimulating factor, which interact with their receptors through binding sites I-III. Site III plays a pivotal role in receptors' dimerization or tetramerization and subsequent activation. Leptin's site III also mediates the formation of an active multimeric complex through its interaction with the IGD (immunoglobulin-like domain) of LEPRs (leptin receptors). Using a sensitive hydrophobic cluster analysis of leptin's and LEPR's sequences, we identified hydrophobic stretches in leptin's A-B loop (amino acids 39-42) and in the N-terminal end of LEPR's IGD (amino acids 325-328) that are predicted to participate in site III and to interact with each other in a beta-sheet-like configuration. To verify this hypothesis, we prepared and purified to homogeneity (as verified by SDS/PAGE, gel filtration and reverse-phase chromatography) several alanine muteins of amino acids 39-42 in human and ovine leptins. CD analyses revealed that those mutations hardly affect the secondary structure. All muteins acted as true antagonists, i.e. they bound LEPR with an affinity similar to the wild-type hormone, had no agonistic activity and specifically inhibited leptin action in several leptin-responsive in vitro bioassays. Alanine mutagenesis of LEPR's IGD (amino acids 325-328) drastically reduced its biological but not binding activity, indicating the importance of this region for interaction with leptin's site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented that the transient FRET signalling occurring upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin's site III.

The effect of pea chloroplast alignment and variation of excitation wavelength on the circularly polarized chlorophyll luminescence.

Circularly polarized luminescence (CPL) is a powerful technique to study the macroorganization of photosynthetic light-harvesting apparatus in vivo and in vitro. It is particularly useful for monitoring environmental stress induced molecular re-organization of thylakoid membranes in green leaves. The current study focuses on two questions which are important to perform and interpret such experiments: how does CPL depend on the excitation wavelength and how on the orientation of the granal thylakoids. CPL and circular dichroism (CD) of pea chloroplasts were complementarily applied when chloroplasts were either in suspension or trapped in a polyacrylamide gel (PAAG) after alignment in a magnetic field. In contrast to the CD spectrum, the CPL signal was found to be independent of the excitation wavelength in both the Soret and the Qy absorption region for chloroplasts in both suspension and PAAG. The improved resolution of luminescence measurements revealed a relatively small negative CPL band in addition to the previously described large positive band. No effect of photoselection upon excitation on the CPL spectra was detected. The CPL intensity at 690 nm at the edge of the granal thylakoids was found to be higher than at the face of the grana suggesting the CPL anisotropy.

Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain.

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.

Chiral macroaggregates of LHCII detected by circularly polarized luminescence in intact pea leaves are sensitive to drought stress.

Circularly polarized chlorophyll luminescence (CPL) was recently shown to be an effective tool for the study of chiral macroaggregate formation of the light-harvesting chlorophyll a/b pigment-protein complexes (LHCIIs) in isolated chloroplasts. The CPL measuring system was modified to study green leaves. Spectral and intensity features of pea leaf CPL signals suggested that CPL indeed detected chiral macroaggregates. The signals were found to depend on the excitation vs emission optical alignment, as well as on the side (adaxial or abaxial) of the leaf. Illumination of attached leaves with either low intensity or strong photoinhibitory light did not affect the chiral macroaggregation status. In contrast, the induction of drought stress in detached leaves led to full disruption of the chiral macroaggregates. The disruption developed gradually during slow dehydration, whereas under fast, heat-stimulated dehydration it was manifested in cooperative diminution of both CPL and photochemical capacity. Simultaneous exposure of the leaves to photoinhibitory illumination and fast dehydration resulted in negative CPL signals. The results demonstrate the potency of CPL as a non-destructive tool for structural studies of LHCII in intact leaves under varying environmental conditions.