Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Strong antenna-enhanced fluorescence of a single light-harvesting complex shows photon anti-bunching
    Wientjes, E. ; Renger, J. ; Curto, A.G. ; Cogdell, R. ; Hulst, N.F. van - \ 2014
    Nature Communications 5 (2014). - ISSN 2041-1723 - 7 p.
    pigment-protein complexes - purple bacterial lh2 - exciton delocalization - molecule fluorescence - energy-transfer - optical antennas - nanoantennas - dynamics - spectroscopy - dna
    The nature of the highly efficient energy transfer in photosynthetic light-harvesting complexes is a subject of intense research. Unfortunately, the low fluorescence efficiency and limited photostability hampers the study of individual light-harvesting complexes at ambient conditions. Here we demonstrate an over 500-fold fluorescence enhancement of lightharvesting complex 2 (LH2) at the single-molecule level by coupling to a gold nanoantenna. The resonant antenna produces an excitation enhancement of circa 100 times and a fluorescence lifetime shortening to B20 ps. The radiative rate enhancement results in a 5.5-fold-improved fluorescence quantum efficiency. Exploiting the unique brightness, we have recorded the first photon antibunching of a single light-harvesting complex under ambient conditions, showing that the 27 bacteriochlorophylls coordinated by LH2 act as a nonclassical single-photon emitter. The presented bright antenna-enhanced LH2 emission is a highly promising system to study energy transfer and the role of quantum coherence at the level of single complexes.
    Exploring the Structure of the 100 Amino-Acid Residue Long N-Terminus of the Plant Antenna Protein CP29
    Shabestari, M.H. ; Wolfs, C.J.A.M. ; Spruijt, R.B. ; Amerongen, H. van; Huber, M. - \ 2014
    Biophysical Journal 106 (2014)6. - ISSN 0006-3495 - p. 1349 - 1358.
    light-harvesting complex - electron-paramagnetic-resonance - comprehensive software package - labeled side-chains - photosystem-ii - distance measurements - conformational-changes - structure prediction - energy-transfer - t4 lysozyme
    The structure of the unusually long (~100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows a-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10-22 and 82-91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane.
    Phosphorescence Imaging of Living Cells with Amino Acid-Functionalized Tris(2-phenylpyridine)iridium(III) Complexes
    Steunenberg, P. ; Ruggi, A. ; Berg, N.S. van den; Buckle, T. ; Kuil, J. ; Leeuwen, F.W.B. van; Velders, A.H. - \ 2012
    Inorganic Chemistry 51 (2012)4. - ISSN 0020-1669 - p. 2105 - 2114.
    cyclometalated iridium complexes - light-emitting-diodes - golgi-apparatus - energy-transfer - emission - ligand - microscopy - ir(iii) - accumulation - derivatives
    A series of nine luminescent cyclometalated octahedral iridium(III) tris(2-phenylpyridine) complexes has been synthesized, functionalized with three different amino acids (glycine, alanine, and lysine), on one, two, or all three of the phenylpyridine ligands. All starting complexes and final compounds have been fully analyzed by one-dimensional (ID) and two-dimensional (2D) NMR spectroscopy, and photophysical data have been obtained for all the mono-, bis-, and tri- substituted iridium(III) complexes. Cellular uptake and localization have been studied with flow cytometry and confocal microscopy, respectively. Confocal experiments demonstrate that all nine substituted iridium(III) complexes show variable uptake in the tumor cells. The monosubstituted iridium(III) complexes give the highest cellular uptake, and the series substituted with lysines shows the highest toxicity. This systematic study of amino acid-functionalized Ir(ppy)(3) complexes provides guidelines for further functionalization and possible implementation of luminescent iridium complexes, for example, in (automated) peptide synthesis or biomarker specific targeting.
    Green-Fluorescent Protein from the Bioluminescent Jellyfish Clytia gregaria Is an Obligate Dimer and Does Not Form a Stable Complex with the Ca2+-Discharged Photoprotein Clytin.
    Malikova, N.P. ; Visser, N.V. ; Hoek, A. van; Skakun, V.V. ; Vysotski, E.S. ; Lee, J. ; Visser, A.J.W.G. - \ 2011
    Biochemistry 50 (2011)20. - ISSN 0006-2960 - p. 4232 - 4241.
    vibrio-fischeri y1 - energy-transfer - correlation spectroscopy - bacterial luciferase - refractive-index - photobacterium-leiognathi - polarized fluorescence - excitation transfer - recombinant obelin - lumazine protein
    Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Fo¨rster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca2+-discharged form that is highly fluorescent (¿max = 506 nm) and its GFP (cgreGFP; ¿max = 500 nm). Ca2+-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca2+-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.
    ATP Changes the Fluorescence Lifetime of Cyan Fluorescent protein via an Interaction with His148
    Borst, J.W. ; Willemse, M. ; Slijkhuis, R. ; Krogt, G. ; Laptenok, S. ; Jalink, K. ; Wieringa, B. ; Fransen, J.A.M. - \ 2010
    PLoS ONE 5 (2010)11. - ISSN 1932-6203 - 7 p.
    energy-transfer - variant - fret - cell - spectroscopy - chromophore - binding - decays
    Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions.
    Efficient light-harvesting by photosystem II requires an optimized protein packing density in grana thylakoids
    Haferkamp, S. ; Haase, W. ; Pascal, A.A. ; Amerongen, H. van; Kirchhoff, H. - \ 2010
    Journal of Biological Chemistry 285 (2010)22. - ISSN 0021-9258 - p. 17020 - 17028.
    energy-transfer - photosynthetic membranes - excitation-energy - higher-plants - charge separation - complexes - organization - chloroplast - model - identification
    A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII
    The chlorosome: a prototype for efficient light harvesting in photosynthesis
    Oostergetel, G. ; Amerongen, H. van; Boekema, E.J. - \ 2010
    Photosynthesis Research 104 (2010)2-3. - ISSN 0166-8595 - p. 245 - 255.
    bacterium chloroflexus-aurantiacus - green sulfur bacteria - pump-probe spectroscopy - chlorobium-tepidum - bacteriochlorophyll-c - energy-transfer - linear dichroism - photoprotection mechanism - pigment organization - antenna protein
    Three phyla of bacteria include phototrophs that contain unique antenna systems, chlorosomes, as the principal light-harvesting apparatus. Chlorosomes are the largest known supramolecular antenna systems and contain hundreds of thousands of BChl c/d/e molecules enclosed by a single membrane leaflet and a baseplate. The BChl pigments are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Their excitation energy flows via a small protein, CsmA embedded in the baseplate to the photosynthetic reaction centres. Chlorosomes allow for photosynthesis at very low light intensities by ultra-rapid transfer of excitations to reaction centres and enable organisms with chlorosomes to live at extraordinarily low light intensities under which no other phototrophic organisms can grow. This article reviews several aspects of chlorosomes: the supramolecular and molecular organizations and the light-harvesting and spectroscopic properties. In addition, it provides some novel information about the organization of the baseplate
    Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs
    Visser, A.J.W.G. ; Laptenok, S. ; Visser, N.V. ; Hoek, A. van; Birch, D.J.S. ; Brochon, J.C. ; Borst, J.W. - \ 2010
    European Biophysics Journal 39 (2010)2. - ISSN 0175-7571 - p. 241 - 253.
    lifetime imaging microscopy - maximum-entropy method - energy-transfer - living cells - polarized fluorescence - flavin fluorescence - lipoamide dehydrogenase - glutathione-reductase - dynamics - photoconversion
    Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive
    Molecular dynamics simulations reveal that AEDANS is an inert fluorescent probe for the study of membrane proteins
    Vos, W.L. ; Schor, M. ; Baumgaertner, A. ; Tieleman, D.P. ; Hemminga, M.A. - \ 2010
    European Biophysics Journal 39 (2010)2. - ISSN 0175-7571 - p. 229 - 239.
    major coat protein - transmembrane alpha-helix - energy-transfer - fret - orientation - conformation - spectroscopy - model - association - bilayers
    Computer simulations were carried out of a number of AEDANS-labeled single cysteine mutants of a small reference membrane protein, M13 major coat protein, covering 60% of its primary sequence. M13 major coat protein is a single membrane-spanning, a-helical membrane protein with a relatively large water-exposed region in the N-terminus. In 10-ns molecular dynamics simulations, we analyze the behavior of the AEDANS label and the native tryptophan, which were used as acceptor and donor in previous FRET experiments. The results indicate that AEDANS is a relatively inert environmental probe that can move unhindered through the lipid membrane when attached to a membrane protein
    Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy
    Berghuis, B.A. ; Spruijt, R.B. ; Koehorst, R.B.M. ; Hoek, A. van; Laptenok, S. ; Oort, B.F. van; Amerongen, H. van - \ 2010
    European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 631 - 638.
    light-harvesting complex - radical-cation formation - plant antenna protein - energy-transfer - photosystem-ii - green plants - escherichia-coli - refractive-index - chlorophyll-a - pump-probe
    A high-throughput Förster resonance energy transfer (FRET) study was performed on the approximately 100 amino acids long N-terminal domain of the photosynthetic complex CP29 of higher plants. For this purpose, CP29 was singly mutated along its N-terminal domain, replacing one-by-one native amino acids by a cysteine, which was labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP9, chlorophylls and xanthophylls. Picosecond fluorescence experiments revealed rapid energy transfer (~20–70 ps) from BODIPY at amino-acid positions 4, 22, 33, 40, 56, 65, 74, 90, and 97 to Chl a molecules in the hydrophobic part of the protein. From the energy transfer times, distances were estimated between label and chlorophyll molecules, using the Förster equation. When the label was attached to amino acids 4, 56, and 97, it was found to be located very close to the protein core (~15 Å), whereas labels at positions 15, 22, 33, 40, 65, 74, and 90 were found at somewhat larger distances. It is concluded that the entire N-terminal domain is in close contact with the hydrophobic core and that there is no loop sticking out into the stroma. Most of the results support a recently proposed topological model for the N-terminus of CP29, which was based on electron-spin-resonance measurements on spin-labeled CP29 with and without its natural pigment content. The present results lead to a slight refinement of that model
    Site-directed spin labeling study of the light-harvesting complex CP29
    Kavalenka, A.A. ; Spruijt, R.B. ; Wolfs, C.J.A.M. ; Strancar, J. ; Croce, R. ; Hemminga, M.A. ; Amerongen, H. van - \ 2009
    Biophysical Journal 96 (2009)9. - ISSN 0006-3495 - p. 3620 - 3628.
    chlorophyll-a/b protein - photosystem-ii subunit - plant antenna protein - n-terminal domain - energy-transfer - conformational-changes - biosystem complexity - membrane-proteins - escherichia-coli - binding protein
    The topology of the long N-terminal domain (100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain
    Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves
    Broess, K. ; Borst, J.W. ; Amerongen, H. van - \ 2009
    Photosynthesis Research 100 (2009)2. - ISSN 0166-8595 - p. 89 - 96.
    light-harvesting-complex - energy-transfer - photosystem-ii - transient absorption - charge separation - chlorophyll-b - green plants - lhcii - time - grana
    This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F 0- and the F m-state. The corresponding average lifetimes are ~250 ps and ~1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of ~500 nm in the focal (xy) plane and 2 ¿m in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed
    Picosecond fluorescence of intact and dissolved PSI-LHCI crystals
    Oort, B.F. van; Amunts, A. ; Borst, J.W. ; Hoek, A. van; Nelson, N. ; Amerongen, H. van; Croce, R. - \ 2008
    Biophysical Journal 95 (2008)12. - ISSN 0006-3495 - p. 5851 - 5861.
    plant photosystem-i - charge separation kinetics - harvesting complex-i - energy-transfer - synechococcus-elongatus - arabidopsis-thaliana - pigment organization - angstrom resolution - antenna complexes - pinacyanol
    Over the last years many crystal structures of photosynthetic pigment-protein complexes have been determined, and used extensively to model spectroscopic results obtained on the same proteins in solution. However, the crystal structure is not necessarily identical to the structure of the protein in solution. Here we studied picosecond fluorescence of Photosystem I-Light Harvesting Complex I (PSI-LHCI), a multisubunit pigment protein complex that catalyzes the first steps of photosynthesis. The ultrafast fluorescence of PSI-LHCI crystals is identical to that of dissolved crystals, but differs considerably from most kinetics presented in literature. In contrast to most studies, the present data can be modeled quantitatively with only 2 compartments: PSI core and LHCI. This yields the rate of charge separation from an equilibrated core (22.5+/-2.5 ps) and rates of excitation energy transfer from LHCI to core (kLC) and vice versa (kCL). The ratio R=kCL/kLC between these rates appears to be wavelength-dependent and scales with the ratio of the absorption spectra of LHCI and core, indicating the validity of a detailed balance relation between both compartments. kLC depends slightly but non systematically on detection wavelength, averaging (9.4+/-4.9 ps)(-1). R ranges from 0.5 (below 690 nm) to around 1.3 above 720 nm.
    Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteriod insensitive 1 receptor oligomerization
    Hink, M.A. ; Shah, K. ; Russinova, E.T. ; Vries, S.C. de; Visser, A.J.W.G. - \ 2008
    Biophysical Journal 94 (2008). - ISSN 0006-3495 - p. 1052 - 1062.
    cross-correlation spectroscopy - protein-protein interactions - photon-counting histogram - live cells - signal-transduction - imaging microscopy - fusion proteins - plasma-membrane - energy-transfer - living cells
    Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes.
    Determination of the excitation migration time in Photosystem II consequences for the membrane organization and charge separation parameters
    Broess, K. ; Trinkunas, G. ; Hoek, A. van; Croce, R. ; Amerongen, H. van - \ 2008
    Biochimica et Biophysica Acta. B, Bioenergetics 1777 (2008)5. - ISSN 0005-2728 - p. 404 - 409.
    light-harvesting complex - transient absorption-spectroscopy - energy-transfer - higher-plants - resolved fluorescence - antenna complex - green plants - thylakoid membrane - mutation analysis - lhcii monomers
    The fluorescence decay kinetics of Photosystem II (PSII) membranes from spinach with open reaction centers (RCs), were compared after exciting at 420 and 484 nm. These wavelengths lead to preferential excitation of chlorophyll (Chl) a and Chl b, respectively, which causes different initial excited-state populations in the inner and outer antenna system. The non-exponential fluorescence decay appears to be 4.3+/-1.8 ps slower upon 484 nm excitation for preparations that contain on average 2.45 LHCII (light-harvesting complex II) trimers per reaction center. Using a recently introduced coarse-grained model it can be concluded that the average migration time of an electronic excitation towards the RC contributes ~23% to the overall average trapping time. The migration time appears to be approximately two times faster than expected based on previous ultrafast transient absorption and fluorescence measurements. It is concluded that excitation energy transfer in PSII follows specific energy transfer pathways that require an optimized organization of the antenna complexes with respect to each other. Within the context of the coarse-grained model it can be calculated that the rate of primary charge separation of the RC is (5.5+/-0.4 ps)(-1), the rate of secondary charge separation is (137+/-5 ps)(-1) and the drop in free energy upon primary charge separation is 826+/-30 cm(-1). These parameters are in rather good agreement with recently published results on isolated core complexes [Y. Miloslavina, M. Szczepaniak, M.G. Muller, J. Sander, M. Nowaczyk, M. Rögner, A.R. Holzwarth, Charge separation kinetics in intact Photosystem II core particles is trap-limited. A picosecond fluorescence study, Biochemistry 45 (2006) 2436-2442].
    FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein
    Nazarov, P.V. ; Koehorst, R.B.M. ; Vos, W.L. ; Apanasovich, V.V. ; Hemminga, M.A. - \ 2007
    Biophysical Journal 92 (2007)4. - ISSN 0006-3495 - p. 1296 - 1305.
    bacteriophage m13 - transmembrane domain - energy-transfer - fluorescence - dynamics - conformation - spectroscopy - modulation - peptides - micelles
    A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall -helical protein conformation from amino acid residues 12¿46, close to the protein conformation in the intact phage.
    Understanding the changes in the circular dichroism of light harvesting complex IIupon varying its pigment composition and organization
    Georgakopoulou, S. ; Zwan, G. van der; Bassi, R. ; Grondelle, R. van; Amerongen, H. van; Croce, R. - \ 2007
    Biochemistry 46 (2007)16. - ISSN 0006-2960 - p. 4745 - 4754.
    a/b-protein complex - lh2 antenna complex - chlorophyll-a - photosystem-ii - higher-plants - rhodopseudomonas-acidophila - chloroplast membranes - mutation analysis - purple bacteria - energy-transfer
    In this work we modeled the circular dichroism (CD) spectrum of LHCII, the main light harvesting antenna of photosystem II of higher plants. Excitonic calculations are performed for a monomeric subunit, taken from the crystal structure of trimeric LHCII from spinach. All of the major features of the CD spectrum above 450 nm are satisfactorily reproduced, and possible orientations of the Chl and carotenoid transition dipole moments are identified. The obtained modeling parameters are used to simulate the CD spectra of two complexes with altered pigment composition: a mutant lacking Chls a 611-612 and a complex lacking the carotenoid neoxanthin. By removing the relevant pigment(s) from the structure, we are able to reproduce their spectra, which implies that the alteration does not disturb the overall structure. The CD spectrum of trimeric LHCII shows a reversed relative intensity of the two negative bands around 470 and 490 nm as compared to monomeric LHCII. The simulations reproduce this reversal, indicating that it is mainly due to interactions between chromophores in different monomeric subunits, and the trimerization does not induce observable changes in the monomeric structure. Our simulated spectrum resembles one of two different trimeric CD spectra reported in literature. We argue that the differences in the experimental trimeric CD spectra are caused by changes in the strength of the monomer-monomer interactions due to the differences in detergents used for the purification of the complexes.
    Interaction of the indole class of vacuolar H+-ATPase inhibitors with lipid bilayers
    Fernandes, F. ; Loura, L. ; Koehorst, R.B.M. ; Dixon, N. ; Kee, T.P. ; Hemminga, M.A. ; Prieto, M. - \ 2006
    Biochemistry 45 (2006)16. - ISSN 0006-2960 - p. 5271 - 5279.
    membrane penetration depth - v-atpase - bone-resorption - fluorescence polarization - selective inhibitor - linear dichroism - energy-transfer - bafilomycin - orientation - tryptophan
    The selective inhibitor of osteoclastic V-ATPase (2Z,4E)-5-(5,6-dichloro-2-indolyi)-2-methoxy-N-(1,2,2,6,6-pentamethylpip eridin-4-yl)-2,4-pentadienamide (SB 242784), member of the indole class of V-ATPase inhibitors, is expected to target the membrane-bound domain of the enzyme. A structural study of the interaction of this inhibitor with the lipidic environment is an essential step in the understanding of the mechanism of inhibition. In this work, a comprehensive study of the relevant features of this interaction was performed. Inhibitor partition coefficients to lipid vesicles as well as its transverse location, orientation (order parameters), and dynamics while bound to bilayers were determined through photophysical techniques, taking advantage of the intrinsic fluorescence of the molecule. To better evaluate the functionally relevant features of SB 242784, a second inhibitor, INH-1, from the same class and having a reduced activity was also examined. It is shown that regarding membrane interaction their properties remain very similar for both molecules, suggesting that the differences in inhibition efficiencies are solely a consequence of the molecular recognition processes within the inhibition site in the V-ATPase.
    Real-time Enzyme Dynamics Illustrated with Fluorescence Spectroscopy of p-Hydroxybenzoate Hydroxylase
    Westphal, A.H. ; Matorin, A. ; Hink, M.A. ; Borst, J.W. ; Berkel, W.J.H. van; Visser, A.J.W.G. - \ 2006
    Journal of Biological Chemistry 281 (2006)16. - ISSN 0021-9258 - p. 11074 - 11081.
    single-molecule kinetics - pseudomonas-fluorescens - conformational dynamics - crystal-structure - energy-transfer - wild-type - catalysis - protein - binding - resolution
    We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair Forster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels shows overlap between its fluorescence spectrum and the absorption spectrum of the FAD prosthetic group in the oxidized state, while the other fluorescent probe does not have this spectral overlap. The spectral overlap strongly diminished when the flavin becomes reduced during catalysis. The donor fluorescence properties can then be used as a sensitive antenna for the flavin redox state. Time-resolved fluorescence experiments on ensembles of labeled PHBH molecules were carried out in the absence and presence of enzymatic turnover. Distinct changes in fluorescence decays of spFRET-active PHBH can be observed when the enzyme is performing catalysis using both substrates p-hydroxybenzoate and NADPH. Single-molecule fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence of a relaxation process (relaxation time of 23 mus) that is related to catalysis. In addition, in both labeled PHBH preparations the number of enzyme molecules reversibly increased during enzymatic turnover indicating that the dimer-monomer equilibrium is affected.
    Spectroscopy and photophysics of self-organized zinc porphyrin nanolayers. 2. Transport properties of singlet excitation
    Donker, H. ; Hoek, A. van; Schaik, W. ; Koehorst, R.B.M. ; Yatskou, M.M. ; Schaafsma, T.J. - \ 2005
    The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 109 (2005)36. - ISSN 1520-6106 - p. 17038 - 17046.
    monte-carlo simulation - energy-transfer - impurity scattering - molecular-crystals - exciton transport - films - fluorescence - systems - phthalocyanine - luminescence
    Exciton diffusion has been studied in 5-25-nm-thick films of zinc tetra-(p-octylphenyl)-porphyrin (ZnTOPP) spin-coated onto quartz slides by intentional doping with quenchers using steady-state as well as time-resolved fluorescence spectroscopy. The fluorescence spectra of the films are very similar to those of solutions, indicating emission from localized exciton states. From the dependence of the fluorescence quenching on the quencher concentration and fluorescence lifetime measurements, the exciton diffusion can be concluded to be quasi-one-dimensional with an exciton diffusion length of 9 ± 3 nm and an intrastack energy-transfer rate constant of 1011-1012 s-1. From fluorescence anisotropy decay measurements, we conclude that neighboring stacks aggregate in a herringbone structure, forming ordered domains that are randomly oriented in the substrate plane. These measurements indicate an interstack energy-transfer rate constant of (7 ± 2) × 1010 s-1
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