Peter J Walla

Max Planck Institute for Biophysical Chemistry, Göttingen, Lower Saxony, Germany

Are you Peter J Walla?

Claim your profile

Publications (46)270.86 Total impact


  • No preview · Article · Dec 2015 · Nature Methods
  • Source

    Full-text · Dataset · Sep 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The Ca(2+) sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin's binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg(2+) completely abrogate synaptotagmin-1 binding to SNAREs regardless of the presence of Ca(2+). Altogether, our data indicate that at physiological ion concentrations Ca(2+)-dependent synaptotagmin-1 binding is confined to PIP2-containing membrane patches in the plasma membrane, suggesting that membrane interaction of synaptotagmin-1 rather than SNARE binding triggers exocytosis of vesicles.
    Full-text · Article · Sep 2015 · Nature Structural & Molecular Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Two-photon fluorescence correlation spectroscopy (2P-FCS) within single dendritic spines of living hippocampal pyramidal neurons was used to resolve various subpopulations of mobile F-actin during activity-dependent structural changes such as potentiation induced spine head growth. Two major classes of mobile F-actin were discovered: very dynamic and about a hundred times less dynamic F-actin. Spine head enlargement upon application of Tetraethylammonium (TEA), a protocol previously used for the chemical induction of long-term potentiation (cLTP) strictly correlated to changes in the dynamics and filament numbers in the different actin filament fractions. Our observations suggest that spine enlargement is governed by a mechanism in which longer filaments are first cut into smaller filaments that cooperate with the second, increasingly dynamic shorter actin filament population to quickly reorganize and expand the actin cytoskeleton within the spine head. This process would allow a fast and efficient spine head enlargement using a major fraction of the actin filament population that was already present before spine head growth.
    Full-text · Article · May 2015 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: It is known that aggregation of isolated light-harvesting complex II (LHCII) in solution results in high fluorescence quenching, reduced chlorophyll fluorescence lifetime, and increased electronic coupling of carotenoid (Car) S1 and chlorophyll (Chl) Qy states, as determined by two-photon studies. It has been suggested that this behavior of aggregated LHCII mimics aspects of non-photochemical quenching processes of higher plants and algae. However, several studies proposed that the minor photosystem II proteins CP24 and CP29 also play a significant role in regulation of photosynthesis. Therefore, we use a simple protocol that allows gradual aggregation also of CP24 and CP29. Similarly, as observed for LHCII, aggregation of CP24 and CP29 also leads to increasing fluorescence quenching and increasing electronic Car S1-Chl Qy coupling. Furthermore, a direct comparison of the three proteins revealed a significant higher electronic coupling in the two minor proteins already in the absence of any aggregation. These differences become even more prominent upon aggregation. A red-shift of the Qy absorption band known from LHCII aggregation was also observed for CP29 but not for CP24. We discuss possible implications of these results for the role of CP24 and CP29 as potential valves for excess excitation energy in the regulation of photosynthetic light harvesting.
    Full-text · Article · Mar 2015 · Photosynthesis Research

  • No preview · Article · Jan 2015 · Biophysical Journal
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fast synchronous neurotransmitter release is triggered by calcium that activates synaptotagmin-1 (syt-1), resulting in fusion of synaptic vesicles with the presynaptic membrane. Syt-1 possesses two Ca(2+)-binding C2 domains that tether membranes via interactions with anionic phospholipids. It is capable of crosslinking membranes and has recently been speculated to trigger fusion by decreasing the gap between them. As quantitative information on membrane gaps is key to understanding general cellular mechanisms, including the role of syt-1, we developed a fluorescence-lifetime based inter-membrane distance ruler using membrane-anchored DNAs of various lengths as calibration standards. Wild-type and mutant data provide evidence that full-length syt-1 indeed regulates membrane gaps: without Ca(2+), syt-1 maintains membranes at distances of ~7-8 nm. Activation with 100 μM Ca(2+) decreases the distance to ~5 nm by binding the C2 domains to opposing membranes, respectively. These values reveal that activated syt-1 adjusts membrane distances to the level that promotes SNARE complex assembly.
    Full-text · Article · Dec 2014 · Nature Communications
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane.
    Full-text · Article · Apr 2014 · Journal of Biological Chemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 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.
    Preview · Article · Apr 2014 · Nature Methods
  • Source

    Preview · Article · Jan 2014 · Biophysical Journal
  • Source
    Christoph-Peter Holleboom · Peter J Walla
    [Show abstract] [Hide abstract]
    ABSTRACT: Many aspects in the regulation of photosynthetic light-harvesting of plants are still quite poorly understood. For example, it is still a matter of debate which physical mechanism(s) results in the regulation and dissipation of excess energy in high light. Many researchers agree that electronic interactions between chlorophylls (Chl) and certain states of carotenoids are involved in these mechanisms. However, in particular, the role of the first excited state of carotenoids (Car S1) is not easily revealed, because of its optical forbidden character. The use of two-photon excitation is an elegant approach to address directly this state and to investigate the energy transfer in the direction Car S1 → Chl. Meanwhile, it has been applied to a large variety of systems starting from simple carotenoid-tetrapyrrole model compounds up to entire plants. Here, we present a systematic summary of the observations obtained by two-photon excitation about Car S1 → Chl energy transfer in systems with increasing complexity and the correlation to fluorescence quenching. We compare these observations directly with the energy transfer in the opposite direction, Chl → Car S1, for the same systems as obtained in pump-probe studies. We discuss what surprising aspects of this comparison led us to the suggestion that quenching excitonic Car-Chl interactions could contribute to the regulation of light harvesting, and how this suggestion can be connected to other models proposed.
    Preview · Article · Apr 2013 · Photosynthesis Research
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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, $${\Phi }_{\hbox{ Coupling }}^{\mathit{C}\mathit{a}\mathit{r}{\mathit{S}}_{\mathbf{1}}-\mathit{C}\mathit{h}\mathit{l}}$$, 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.
    Preview · Article · Mar 2013 · Proceedings of the National Academy of Sciences
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The regulation of light-harvesting in photosynthesis under conditions of varying solar light irradiation is essential for the survival and fitness of plants and algae. It has been proposed that rearrangements of protein distribution in the stacked grana region of thylakoid membranes connected to changes in the electronic pigment-interaction play a key role for this regulation. In particular, carotenoid-chlorophyll interactions seem to be crucial for the down-regulation of photosynthetic light-harvesting. So far, it has been difficult to determine the influence of the dense protein packing found in native photosynthetic membrane on these interactions. We investigated the changes of the electronic couplings between carotenoids and chlorophylls and the quenching in grana thylakoids of varying protein packing density by two-photon spectroscopy, conventional chlorophyll fluorometry, low-temperature fluorescence spectroscopy, and electron micrographs of freeze-fracture membranes. We observed an increasing carotenoid-chlorophyll coupling and fluorescence quenching with increasing packing density. Simultaneously, the antennas size and excitonic connectivity of Photosystem II increased with increasing quenching and carotenoid-chlorophyll coupling whereas isolated, decoupled LHCII trimers decreased. Two distinct quenching data regimes could be identified that show up at different protein packing densities. In the regime corresponding to higher protein packing densities, quenching is strongly correlated to carotenoid-chlorophyll interactions whereas in the second regime, a weak correlation is apparent with low protein packing densities. Native membranes are in the strong-coupling data regime. Consequently, PSII and LHCII in grana membranes of plants are already quenched by protein crowding. We concluded that this ensures efficient electronic connection of all pigment-protein complexes for intermolecular energy transfer to the reaction centers and allows simultaneously sensitive regulation of light harvesting by only small changes in the protein packaging.
    Full-text · Article · Mar 2013 · The Journal of Physical Chemistry B
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Preview · Article · Jul 2012 · Journal of Biological Chemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Full-text · Article · Jun 2012 · Science
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 Ca(2+) 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 Ca(2+)-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 Ca(2+)-binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca(2+)-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.
    Full-text · Article · Jun 2012 · Proceedings of the National Academy of Sciences
  • [Show abstract] [Hide abstract]
    ABSTRACT: In two recent studies, energy transfer was reported in certain phthalocyanine-carotenoid dyads between the optically forbidden first excited state of carotenoids (Car S(1)) and phthalocyanines (Pcs) in the direction Pc → Car S(1) (Kloz et al., J Am Chem Soc 133:7007-7015, 2011) as well as in the direction Car S(1) → Pc (Liao et al., J Phys Chem A 115:4082-4091, 2011). In this article, we show that the extent of this energy transfer in both directions is closely correlated in these dyads. This correlation and the additional observation that Car S(1) is instantaneously populated after Pc excitation provides evidence that in these compounds excitonic interactions can occur. Besides pure energy transfer and electron transfer, this is the third type of tetrapyrrole-carotenoid interaction that has been shown to occur in these model compounds and that has previously been proposed as a photosynthetic regulation mechanism. We discuss the implications of these models for photosynthetic regulation. The findings are also discussed in the context of a model in which both electronic states are disordered and in which the strength of the electronic coupling determines whether energy transfer, excitonic coupling, or electron transfer occurs.
    No preview · Article · Sep 2011 · Photosynthesis Research
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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).
    No preview · Article · Jun 2011 · Journal of Biological Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: Electronic interactions between the first excited states (S(1)) of carotenoids (Car) of different conjugation lengths (8-11 double bonds) and phthalocyanines (Pc) in different Car-Pc dyad molecules were investigated by two-photon spectroscopy and compared with Car S(1)-chlorophyll (Chl) interactions in photosynthetic light harvesting complexes (LHCs). The observation of Chl/Pc fluorescence after selective two-photon excitation of the Car S(1) state allowed sensitive monitoring of the flow of energy between Car S(1) and Pc or Chl. It is found that two-photon excitation excites to about 80% to 100% exclusively the carotenoid state Car S(1) and that only a small fraction of direct tetrapyrrole two-photon excitation occurs. Amide-linked Car-Pc dyads in tetrahydrofuran demonstrate a molecular gear shift mechanism in that effective Car S(1) → Pc energy transfer is observed in a dyad with 9 double bonds in the carotenoid, whereas in similar dyads with 11 double bonds in the carotenoid, the Pc fluorescence is strongly quenched by Pc → Car S(1) energy transfer. In phenylamino-linked Car-Pc dyads in toluene extremely large electronic interactions between the Car S(1) state and Pc were observed, particularly in the case of a dyad in which the carotenoid contained 10 double bonds. This observation together with previous findings in the same system provides strong evidence for excitonic Car S(1)-Pc Q(y) interactions. Very similar results were observed with photosynthetic LHC II complexes in the past, supporting an important role of such interactions in photosynthetic down-regulation.
    No preview · Article · Mar 2011 · The Journal of Physical Chemistry A
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    No preview · Article · Nov 2010 · The Journal of Physical Chemistry B

Publication Stats

1k Citations
270.86 Total Impact Points

Institutions

  • 2006-2015
    • Max Planck Institute for Biophysical Chemistry
      • Group of Biomolecular Spectroscopy and Single Molecule Detection
      Göttingen, Lower Saxony, Germany
  • 2004-2015
    • Technische Universität Braunschweig
      • • Zoological Institute
      • • Institute of physical and theoretical chemistry
      Brunswyck, Lower Saxony, Germany
  • 1999-2013
    • University of California, Berkeley
      • Department of Chemistry
      Berkeley, California, United States
  • 2011
    • Arizona State University
      • The Center for Bioenergy and Photosynthesis
      Phoenix, Arizona, United States