Peter J. Walla

On the regulation of photosynthesis by excitonic interactions between carotenoids and chlorophylls.

Selective 2-photon excitation (TPE) of carotenoid dark states, Car S1, shows that in the major light-harvesting complex of photosystem II (LHCII), the extent of electronic interactions between carotenoid dark states (Car S1) and chlorophyll (Chl) states, φCouplingCar S1−Chl, correlates linearly with chlorophyll fluorescence quenching under different experimental conditions. Simultaneously, a linear correlation between both Chl fluorescence quenching and φCouplingCar S1−Chl with the intensity of red-shifted bands in the Chl Qy and carotenoid absorption was also observed. These results suggest quenching excitonic Car S1−Chl states as origin for the observed effects. Furthermore, real time measurements of the light-dependent down- and up-regulation of the photosynthetic activity and φCouplingCar S1−Chl in wild-type and mutant (npq1, npq2, npq4, lut2 and WT+PsbS) Arabidopsis thaliana plants reveal that also in vivo the quenching parameter NPQ correlates always linearly with the extent of electronic Car S1–Chl interactions in any adaptation status. Our in vivo measurements with Arabidopsis variants show that during high light illumination, φCouplingCar S1−Chl depends on the presence of PsbS and zeaxanthin (Zea) in an almost identical way as NPQ. In summary, these results provide clear evidence for a very close link between electronic Car S1–Chl interactions and the regulation of photosynthesis. These findings support a photophysical mechanism in which short-living, low excitonic carotenoid–chlorophyll states serve as traps and dissipation valves for excess excitation energy.

Membrane Fusion Intermediates via Directional and Full Assembly of the SNARE Complex

No More Fusion Confusion Biophysical models explain membrane fusion as a sequence of steps—including membrane contact, formation of a fusion stalk (merger of proximal monolayers), development of contact between distal monolayers that may or may not expand (hemifusion), and, finally, rupture of this diaphragm resulting in the opening of a fusion pore. Biological membrane fusion reactions are often driven by so-called SNARE proteins. By using a reconstituted membrane fusion system, Hernandez et al. (p. 1581, published online 31 May) have now been able to correlate precisely the states of SNARE zippering with intermediate structures along the fusion pathway. The results suggest that a tightly docked state, with a membrane distance so close that no proteins fit in between them, represents a critical fusion intermediate as a consequence of SNARE zippering. This intermediate is incompatible with a SNARE-driven stalk or with a ringlike arrangement of SNAREs depicted in most current models of membrane fusion. During vesicle membrane fusion, straining of lipids at the edges of an extended contact zone may initiate fusion. Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction, we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.

Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies

SNARE proteins mediate membrane fusion in eukaryotic cells. They contain conserved SNARE motifs that are usually located adjacent to a C‐terminal transmembrane domain. SNARE motifs spontaneously assemble into four helix bundles, with each helix belonging to a different subfamily. Liposomes containing SNAREs spontaneously fuse with each other, but it is debated how the SNAREs are distributed between the membranes. Here, we report that the SNAREs mediating homotypic fusion of early endosomes fuse liposomes in five out of seven possible combinations, in contrast to previously studied SNAREs involved in heterotypic fusion events. The crystal structure of the early endosomal SNARE complex resembles that of the neuronal and late endosomal complexes, but differs in surface side‐chain interactions. We conclude that homotypic fusion reactions may proceed with multiple SNARE topologies, suggesting that the conserved SNARE structure allows for flexibility in the initial interactions needed for fusion.

Direct interaction of the major light-harvesting complex II and PsbS in nonphotochemical quenching

The photosystem II (PSII) subunit S (PsbS) plays a key role in nonphotochemical quenching, a photoprotective mechanism for dissipation of excess excitation energy in plants. The precise function of PsbS in nonphotochemical quenching is unknown. By reconstituting PsbS together with the major light-harvesting complex of PSII (LHC-II) and the xanthophyll zeaxanthin (Zea) into proteoliposomes, we have tested the individual contributions of PSII complexes and Zea to chlorophyll (Chl) fluorescence quenching in a membrane environment. We demonstrate that PsbS is stable in the absence of pigments in vitro. Significant Chl fluorescence quenching of reconstituted LHC-II was observed in the presence of PsbS and Zea, although neither Zea nor PsbS alone was sufficient to induce the same quenching. Coreconstitution with PsbS resulted in the formation of LHC-II/PsbS heterodimers, indicating their direct interaction in the lipid bilayer. Two-photon excitation measurements on liposomes containing LHC-II, PsbS, and Zea showed an increase of electronic interactions between carotenoid S1 and Chl states, , that correlated directly with Chl fluorescence quenching. These findings are in agreement with a carotenoid-dependent Chl fluorescence quenching by direct interactions of LHCs of PSII with PsbS monomers.

Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS

Neuronal exocytosis is mediated by the SNARE proteins synaptobrevin 2/VAMP, syntaxin 1A, and SNAP-25A. While it is well-established that these proteins mediate membrane fusion after reconstitution in artificial membranes, it has so far been difficult to monitor intermediate stages of the reaction. Using a confocal two-photon setup, we applied fluorescence cross-correlation spectroscopy (FCCS) and fluorescence lifetime analysis to discriminate between docking and fusion of liposomes. We show that liposome populations that are either non-interacting, or are undergoing docking and fusion, as well as multiple interactions can be quantitatively discriminated without the need for immobilizing the lipid bilayers. When liposomes containing a stabilized syntaxin 1A/SNAP-25A complex were mixed with liposomes containing synaptobrevin 2, we observed that rapid docking precedes fusion. Accordingly, docked intermediates accumulated in the initial phase of the reaction. Furthermore, rapid formation of multiple docked states was observed with on average four liposomes interacting with each other. When liposomes of different sizes were compared, only the rate of lipid mixing depended on the liposome size but not the rate of docking. Our results show that under appropriate conditions a docked state, mediated by trans-SNARE interactions, can be isolated that constitutes an intermediate in the fusion pathway.

Cis- and trans-membrane interactions of synaptotagmin-1

In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca2+ triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in cis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca2+-dependent and -independent cis-binding and trans-tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca2+-binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca2+-dependent cis-binding prevents trans-interactions if the cis-membrane contains 12–20% anionic phospholipids. Similarly, no trans-interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans-interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between cis- and trans-binding, which may play an important modulatory role in synaptic transmission.

Correlation of Car S1 -> Chl with Chl -> Car S1 energy transfer supports the excitonic model in quenched light harvesting complex II.

Recently, excitonic carotenoid-chlorophyll interactions have been proposed as a simple but effective model for the down-regulation of photosynthesis in plants. The model was proposed on the basis of quenching-correlated electronic carotenoid-chlorophyll interactions (Car S(1) → Chl) determined by Car S(1) two-photon excitation and red-shifted absorption bands. However, if excitonic interactions are indeed responsible for this effect, a simultaneous correlation of quenching with increased energy transfer in the opposite direction, Chl Q(y) → Car S(1), should be observed. Here we present a systematic study on the correlation of Car S(1) → Chl and Chl → Car S(1) energy transfer with the occurrence of red-shifted bands and quenching in isolated LHCII. We found a direct correlation between all four phenomena, supporting our conclusion that excitonic Car S(1)-Chl interactions provide low-lying states serving as energy traps and dissipative valves for excess excitation energy.

Methylation of K9 in histone H3 directs alternative modes of highly dynamic interaction of heterochromatin protein hHP1β with the nucleosome.

Background: Chromatin-HP1 (heterochromatin protein 1) interaction is crucial for heterochromatin assembly. Results: hHP1β uses alternative interfaces to bind nucleosomes depending on histone 3 methylation within a highly dynamic complex. Conclusion: hHP1β explores chromatin for sites of methyl-mark enrichment where it can bind histone 3 tails from adjacent nucleosomes. Significance: We provide a conceptual framework to understand the molecular basis of dynamic interactions regulated by histone modification. Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1β to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1β with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1β-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin.

Photoprotection in plants involves a change in lutein 1 binding domain in the major light-harvesting complex of photosystem II

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S1) performed on LHCII gels shows that the extent of electronic interactions between Car S1 and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)2 image nanoareas can, in combination with application of the SPEED (sparsity penalty-enhanced estimation by demodulation) deconvolution algorithm, provide subdiffraction resolution (super resolution by polarization demodulation, SPoD). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, ExPAN). This shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. Our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.