Publications
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McClelland A, Demidov A, Benabbas A, et al., 2010, Investigation of excited state proton transfer in green fluorescent protein, ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, Vol: 240, ISSN: 0065-7727
McClelland A, Demidov A, Benabbas A, et al., 2010, Direct Observations of Low Frequency Vibrational Coherences in wt-GFP, XXII INTERNATIONAL CONFERENCE ON RAMAN SPECTROSCOPY, Pages: 674-675
McClelland A, Demidov A, Benabbas A, et al., 2010, Investigation of excited state proton transfer in green fluorescent protein, Abstracts of Papers of the American Chemical Society
Wilderen LJGWV, Clark IP, Towrie M, et al., 2009, Mid-infrared picosecond pump-dump-probe and pump-repump-probe experiments to resolve a ground-state intermediate in cyanobacterial phytochrome Cph1, J Phys Chem B, Vol: 113, Pages: 16354-16364
Multipulse picosecond mid-infrared spectroscopy has been used to study photochemical reactions of the cyanobacterial phytochrome photoreceptor Cph1. Different photophysical schemes have been discussed in the literature to describe the pathways after photoexcitation, particularly, to identify reaction phases that are linked to photoisomerisation and electronic decay in the 1566-1772 cm(-1) region that probes C=C and C=O stretching modes of the tetrapyrrole chromophore. Here, multipulse spectroscopy is employed, where, compared to conventional visible pump-mid-infrared probe spectroscopy, an additional visible pulse is incorporated that interacts with populations that are evolving on the excited- and ground-state potential energy surfaces. The time delays between the pump and the dump pulse are chosen such that the dump pulse interacts with different phases in the reaction process. The pump and dump pulses are at the same wavelength, 640 nm, and are resonant with the Pr ground state as well as with the excited state and intermediates. Because the dump pulse additionally pumps the remaining, partially recovered, and partially oriented ground-state population, theory is developed for estimating the fraction of excited-state molecules. The calculations take into account the model-dependent ground-state recovery fraction, the angular dependence of the population transfer resulting from the finite bleach that occurs with linearly polarized intense femtosecond optical excitation, and the partially oriented population for the dump field. Distinct differences between the results from the experiments that use a 1 or a 14 ps dump time favor a branching evolution from S1 to an excited state or reconfigured chromophore and to a newly identified ground-state intermediate (GSI). Optical dumping at 1 ps shows the instantaneous induced absorption of a delocalized C=C stretching mode at 1608 cm(-1), where the increased cross section is associated with the electronic ground-state struc
van Thor JJ, 2009, Photoreactions and dynamics of the green fluorescent protein, Chem Soc Rev, Vol: 38, Pages: 2935-2950
The wild type green fluorescent protein (GFP) from Aequorea victoria has been extensively investigated with a strong focus on the photochemistry and structural dynamics that are linked with its diverse activities. GFP combines a number of remarkable, and some unique, features that are still intensely researched both experimentally and theoretically. The protein environment effectively inhibits deactivation pathways that are dominant in the isolated chromophore and is therefore responsible for the bright fluorescence. Its p-hydroxybenzylidene-imidazolidinone chromophore acts as a photoacid, and optical excitation triggers ultrafast proton transfer reactions in the active site. The microscopic details of the proton transfer mechanism through a hydrogen bonding network are discussed in this critical review. This property of the wild type GFP has provided the opportunity to characterise the role of the specific protein environment in the proton transfer reactions in comparison to photoacid reactions in the condensed phase. In addition, GFP displays a photochromic side reaction that is uniquely caused by electron transfer from a buried anionic glutamic acid to the optically excited chromophore. This phototransformation property has also been exploited in high resolution fluorescence microscopy techniques. The discussion in this review is extended to include vibrational spectroscopy and structural dynamics (106 references).
van Thor JJ, Ronayne KL, Towrie M, et al., 2009, Deriving molecular information from photoselection experiments of the green fluorescent protein using intense femtosecond pulses, CCLRC Central Laser Facility Annual Report 2007-2008, Publisher: CCLRC
van Thor JJ, Ronayne KL, Towrie M, et al., 2008, Balance between parallel ultrafast excited state proton transfer reactions in GFP has a structural origin., Biophys J, Vol: 95, Pages: 1902-1912
The fluorescence photocycle of the green fluorescent protein is functionally dependent on the specific structural protein environment. A direct relationship between equilibrium protein side-chain conformation of glutamate 222 and reactivity is established, particularly the rate of ultrafast proton transfer reactions in the fluorescence photocycle. We show that parallel transformations in the photocycle have a structural origin, and we report on the vibrational properties of responsive amino acids on an ultrafast timescale. Blue excitation of GFP drives two parallel, excited-state deuteron transfer reactions with 10 ps and 75 ps time constants to the buried carboxylic acid side chain of glutamate 222 via a hydrogen-bonding network. Assignment of 1456 cm(-1) and 1441 cm(-1) modes to nu(sym) and assignment of 1564 cm(-1) and 1570 cm(-1) features to nu(asym) of E222 in the 10 ps and 75 ps components, respectively, was possible from the analysis of the transient absorption data of an E222D mutant and was consistent with photoselection measurements. In contrast to the wild-type, measurements of E222D can be described with only one difference spectrum, with the nu(sym) mode at 1435 cm(-1) and the nu(asym) mode at 1567 cm(-1), also correlating a large Deltanu(asym-sym) with slow excited-state proton transfer kinetics. Density Functional Theory calculations and published model compound and theoretical studies relate differences in Deltanu(asym-sym) to the strength and number of hydrogen-bonding interactions that are detected via equilibrium geometry and COO- stretching frequency differences of the carboxylate. The correlation of photocycle kinetics with side-chain conformation of the acceptor suggests that proton transfer from S205 to E222 controls the rate of the overall excited-state proton transfer process, which is consistent with recent theoretical predictions. Photoselection measurements show agreement for localized C=O vibrations of chromophore, Q69, and E222 with Den
van Thor JJ, Ronayne KL, Towrie M, 2007, Formation of the early photoproduct Lumi-R of cyanobacterial phytochrome Cph1 observed by ultrafast mid-infrared spectroscopy, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 129, Pages: 126-132, ISSN: 0002-7863
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- Citations: 82
van Thor JJ, Mackeen M, Kuprov I, et al., 2006, Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1, BIOPHYSICAL JOURNAL, Vol: 91, Pages: 1811-1822, ISSN: 0006-3495
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- Citations: 43
van Thor JJ, Sage JT, 2006, Charge transfer in green fluorescent protein, PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, Vol: 5, Pages: 597-602, ISSN: 1474-905X
van Thor JJ, Towrie M, Ronayne K, et al., 2006, Ultrafast and low barrier motions in the Photoreactions of the Green Fluorescent Protein, Conference on Genetically Engineered Probes for Biomedical Applications, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
van Thor JJ, Zanetti G, Ronayne K, et al., 2006, Picosecond time resolved infrared absorption measurements reporting on the structural events in the photocycle of Green Fluorescent Protein, CCLRC Central Laser Facility Annual Report 2004-2005, Publisher: CCLRC
van Thor JJ, Fisher N, Rich PR, 2005, Assignments of the Pfr-Pr FTIR difference spectrum of cyanobacterial phytochrome Cph1 using <SUP>15</SUP>N and <SUP>13</SUP>C isotopically labeled phycocyanobilin chromophore, JOURNAL OF PHYSICAL CHEMISTRY B, Vol: 109, Pages: 20597-20604, ISSN: 1520-6106
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- Citations: 48
van Thor JJ, Georgiev GY, Towrie M, et al., 2005, Ultrafast and low barrier motions in the photoreactions of the green fluorescent protein, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 280, Pages: 33652-33659, ISSN: 0021-9258
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- Citations: 56
van Thor JJ, Zanetti G, Ronayne KL, et al., 2005, Structural events in the photocycle of green fluorescent protein, JOURNAL OF PHYSICAL CHEMISTRY B, Vol: 109, Pages: 16099-16108, ISSN: 1520-6106
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- Citations: 61
Kennis JTM, Larsen DS, van Stokkum IHM, et al., 2004, Uncovering the hidden ground state of green fluorescent protein, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 101, Pages: 17988-17993, ISSN: 0027-8424
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- Citations: 125
Kennis JTM, Larsen DS, van Stokkum IHM, et al., 2004, Uncovering the hidden ground state of green fluorescent protein, 48th Annual Meeting of the Biophysical Society, Publisher: BIOPHYSICAL SOCIETY, Pages: 168A-168A, ISSN: 0006-3495
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- Citations: 3
Hendriks J, Gensch T, Hviid L, et al., 2002, Transient exposure of hydrophobic surface in the photoactive yellow protein monitored with Nile red, BIOPHYSICAL JOURNAL, Vol: 82, Pages: 1632-1643, ISSN: 0006-3495
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- Citations: 86
van Thor JJ, Gensch T, Hellingwerf KJ, et al., 2002, Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222, NATURE STRUCTURAL BIOLOGY, Vol: 9, Pages: 37-41, ISSN: 1072-8368
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- Citations: 182
van Thor JJ, Hellingwerf KJ, 2002, Fluorescence resonance energy transfer (FRET) applications using green fluorescent protein. Energy transfer to the endogenous chromophores of phycobilisome light-harvesting complexes., Methods Mol Biol, Vol: 183, Pages: 101-119, ISSN: 1064-3745
van Thor JJ, Gensch T, Hellingwerf KJ, et al., 2002, Phototransformation of the wildtype green fluorescent protein with UVV- and Visible light leads to decarboxylation of glutamate 222, Bioluminescence & chemiluminescence, Editors: Stanley, Kricka, Publisher: World Scientific Pub Co Inc, ISBN: 9789812381569
This volume presents a compilation of the latest developments from key experts and leading-edge researchers in this area.
van Thor JJ, Borucki B, Crielaard W, et al., 2001, Light-induced proton release and proton uptake reactions in the cyanobacterial phytochrome Cph1, BIOCHEMISTRY, Vol: 40, Pages: 11460-11471, ISSN: 0006-2960
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- Citations: 100
van Thor JJ, Jeanjean R, Haveaux M, et al., 2000, Salt shock-inducible photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of ferredoxin:NADP(+) reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous N-terminal domain., Biochim Biophys Acta, Vol: 1457, Pages: 129-144
Relative to ferredoxin:NADP(+) reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terminus. The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae. The phenotypic consequences of the genetic removal of this domain from the petH gene, which encodes FNR, have been studied in Synechocystis PCC 6803. The in frame deletion of 75 residues at the amino-terminus, rendered chloroplast length FNR enzyme with normal functionality in linear photosynthetic electron transfer. Salt shock correlated with increased abundance of petH mRNA in the wild-type and mutant alike. The truncation stopped salt stress-inducible increase of Photosystem I-dependent cyclic electron flow. Both photoacoustic determination of the storage of energy from Photosystem I specific far-red light, and the re-reduction kinetics of P700(+), suggest lack of function of the truncated FNR in the plastoquinone-cytochrome b(6)f complex reductase step of the PS I-dependent cyclic electron transfer chain. Independent gold-immunodecoration studies and analysis of FNR distribution through activity staining after native polyacrylamide gelelectrophoresis showed that association of FNR with the thylakoid membranes of Synechocystis PCC 6803 requires the presence of the extended amino-terminal domain of the enzyme. The truncated DeltapetH gene was also transformed into a NAD(P)H dehydrogenase (NDH1) deficient mutant of Synechocystis PCC 6803 (strain M55) (T. Ogawa, Proc. Natl. Acad. Sci. USA 88 (1991) 4275-4279). Phenotypic characterisation of the double mutant supported our conclusion that both the NAD(P)H dehydrogenase complex and FNR contribute independently to the quinone cytochrome b(6)f reductase step in PS I-dependent cyclic electron transfer. The distribution, binding properties and function of FNR in the model cyanobacterium Synechocystis PCC 6803 will be discussed.
Hellingwerf KJ, van Thor JJ, Hendriks J, et al., 2000, Nanotechnologie, Nanotechnologie, Editors: Wolde, Jongeling, ISBN: 9789073035874
van Thor JJ, Gruters OW, Matthijs HC, et al., 1999, Localization and function of ferredoxin:NADP(+) reductase bound to the phycobilisomes of Synechocystis, EMBO Journal, Vol: 18, Pages: 4128-4136
Each phycobilisome complex of the cyanobacterium Synechocystis PCC 6803 binds approximately 2.4 copies of ferredoxin:NADP(+) reductase (FNR). A mutant of this strain that carries an N-terminally truncated version of the petH gene, lacking the 9 kDa domain of FNR that is homologous to the phycocyanin-associated linker polypeptide CpcD, assembles phycobilisome complexes that do not contain FNR. Phycobilisome complexes, consisting of the allophycocyanin core and only the core-proximal phycocyanin hexamers from mutant R20, do contain a full complement of FNR. Therefore, the binding site of FNR in the phycobilisomes is not the core-distal binding site that is occupied by CpcD, but in the core-proximal phycocyanin hexamer. Phycobilisome complexes of a mutant expressing a fusion protein of the N-terminal domain of FNR and green fluorescent protein (GFP) contain this fusion protein in tightly bound form. Calculations of the fluorescence resonance energy transfer (FRET) characteristics between GFP and acceptors in the phycobilisome complex indicate that their donor-acceptor distance is between 3 and 7 nm. Fluorescence spectroscopy at 77K and measurements in intact cells of accumulated levels of P700(+) indicate that the presence of FNR in the phycobilisome complexes does not influence the distribution of excitation energy of phycobilisome-absorbed light between photosystem II and photosystem I, and also does not affect the occurrence of 'light-state transitions'
van Thor JJ, Geerlings TH, Matthijs HC, et al., 1999, Kinetic evidence for the PsaE-dependent transient ternary complex photosystem I/Ferredoxin/Ferredoxin:NADP(+) reductase in a cyanobacterium, Biochemistry, Vol: 38, Pages: 12735-12746
A mutant of Synechocystis PCC 6803, deficient in psaE, assembles photosystem I reaction centers without the PsaE subunit. Under conditions of acceptor-side rate-limited photoreduction assays in vitro (with 15 microM plastocyanin included), using 100 nM ferredoxin:NADP(+) reductase (FNR) and either Synechocystis flavodoxin or spinach ferredoxin, lower rates of NADP(+) photoreduction were measured when PsaE-deficient membranes were used, as compared to the wild type. This effect of the psaE mutation proved to be due to a decrease of the apparent affinity of the photoreduction assay system for the reductase. In the psaE mutant, the relative petH (encoding FNR) expression level was found to be significantly increased, providing a possible explanation for the lack of a phenotype (i.e., a decrease in growth rate) that was expected from the lower rate of linear electron transport in the mutant. A kinetic model was constructed in order to simulate the electron transfer from reduced plastocyanin to NADP(+), and test for possible causes for the observed change in affinity for FNR. The numerical simulations predict that the altered reduction kinetics of ferredoxin, determined for the psaE mutant [Barth, P., et al., (1998) Biochemistry 37, 16233-16241], do not significantly influence the rate of linear electron transport to NADP(+). Rather, a change in the dissociation constant of ferredoxin for FNR does affect the saturation profile for FNR. We therefore propose that the PsaE-dependent transient ternary complex PSI/ferredoxin/FNR is formed during linear electron transport. Using the yeast two-hybrid system, however, no direct interaction could be demonstrated in vivo between FNR and PsaE fusion proteins
van Thor JJ, Mullineaux CW, Matthijs HC, et al., 1998, Light harvesting and state transitions in cyanobacteria, Botanica Acta, Vol: 111, Pages: 430-443
van Thor JJ, Hellingwerf KJ, Matthijs HC, 1998, Characterization and transcriptional regulation of the Synechocystis PCC 6803 petH gene, encoding ferredoxin-NADP+ oxidoreductase: involvement of a novel type of divergent operator, Plant Mol Biol, Vol: 36, Pages: 353-363
The petH gene, encoding ferredoxin-NADP+ oxidoreductase (FNR), has been characterised in the unicellular cyanobacterium Synechocystis PCC 6803. Its product, FNR, was heterologously produced and functionally characterized. The start-site of the monocystronic petH transcript was mapped 523 bp upstream of the predicted PetH initiation codon, resulting in an unusually large 5'-untranslated region. The 5' end of the petH transcript is situated within the open reading frame of phosphoribulokinase (encoded by prk), which is transcribed in opposite orientation with respect to petH. The transcription start site of the prk transcript was mapped 219 bp upstream of the initiation codon, resulting in a 223 bp antisense region between both transcripts. Under many conditions the expression of both genes (i.e. petH and prk) is co-regulated symmetrically at the transcriptional level, as was concluded from both northern hybridization experiments and from primer extension analyses; it became uncoupled, however, when specifically petH expression was stimulated, independent of prk expression, by stressing the Synechocystis cells with high salt concentrations. A model for a new type of bidirectional operator, regulating the expression of petH and prk, is proposed.
van Thor JJ, Pierik AJ, Nugteren-Roodzandt I, et al., 1998, Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy., Biochemistry, Vol: 37, Pages: 16915-16921
Green Fluorescent Protein (GFP) is a bioluminescence protein from the jelly fish Aequorea victoria. It can exist in at least two spectroscopically distinct states: GFP395 and GFP480, with peak absorption at 395 and 480 nm, respectively, presumably resulting from a change in the protonation state of the phenolic ring of its chromophore. When GFP is formed upon heterologous expression in Escherichia coli, its chromophore is mainly present as the neutral species. UV and visible light convert (the chromophore of) GFP quantitatively from this neutral- into the anionic form. On the basis of X-ray diffraction, it was recently proposed (Brejc, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2306-2311; Palm, G. J. et al. (1997) Nat. Struct. Biol. 4, 361-365) that the carboxylic group of Glu222 functions as the proton acceptor of the chromophore of GFP, during the transition from the neutral form (i.e., GFP395) to the ionized form (GFP480). However, X-ray crystallography cannot detect protons directly. The results of FTIR difference spectroscopy, in contrast, are highly sensitive to changes in the protonation state between two conformations of a protein. Here we report the first characterization of GFP, and its photoconversion, with FTIR spectroscopy. Our results clearly show the change in protonation state of the chromophore upon photoconversion. However, they do not provide indications for a change of the protonation state of a glutamate side chain between the states GFP395 and GFP480, nor for an isomerization of the double bond that forms part of the link between the two rings of the chromophore.
Springael D, van Thor JJ, Goorissen H, et al., 1996, RP4::Mu3A-mediated in vivo cloning and transfer of a chlorobiphenyl catabolic pathway, Microbiology, Vol: 142, Pages: 3282-3293
Chromosomal DNA fragments encoding the ability to utilize biphenyl as sole carbon source (Bph+) were mobilized by means of plasmid RP4::Mu3A from strain JB1 (tentatively identified as Burkholderia sp.) to Alcaligenes eutrophus CH34 at a frequency of 10(-3) per transferred plasmid. The mobilized DNA integrated into the recipient chromosome or was recovered as catabolic prime plasmids. Three Bph+ prime plasmids were transferred from A. eutrophus to Escherichia coli and back to A. eutrophus without modification of the phenotype. The transferred Bph+ DNA segments allowed metabolism of biphenyl, 2-, 3- and 4-chlorobiphenyl, and diphenylmethane. Genes involved in biphenyl degradation were identified on the prime plasmids by DNA-DNA hybridization and by gene cloning. Bph+ prime plasmids were transferred to Burkholderia cepacia, Pseudomonas aeruginosa, Comamonas testosteroni and A. eutrophus and the catabolic genes were expressed in those hosts. Transfer of the plasmid to the 3-chlorobenzoate-degrading bacterium Pseudomonas sp. B13 allowed the recipient to mineralize 3-chlorobiphenyl. Other catabolic prime plasmids were obtained from JB1 by selection on m-hydroxybenzoate and tyrosine as carbon sources. 16S rRNA sequence data demonstrated that the in vivo transfer of bph was achieved between bacteria belonging to two different branches of the beta-Proteobacteria.
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