Search PubMed⌕ Search

Biomedical subjects

K J Hellingwerf

Publications and source records attributed to K J Hellingwerf.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effects of limited aeration and of the ArcAB system on intermediary pyruvate catabolism in Escherichia coli.

The capacity of Escherichia coli to adapt its catabolism to prevailing redox conditions resides mainly in three catabolic branch points involving (i) pyruvate formate-lyase (PFL) and the pyruvate dehydrogenase complex (PDHc), (ii) the exclusively fermentative enzymes and those of the Krebs cycle, and (iii) the alternative terminal cytochrome bd and cytochrome bo oxidases. A quantitative analysis of the relative catabolic fluxes through these pathways is presented for steady-state glucose-limited chemostat cultures with controlled oxygen availability ranging from full aerobiosis to complete anaerobiosis. Remarkably, PFL contributed significantly to the catabolic flux under microaerobic conditions and was found to be active simultaneously with PDHc and cytochrome bd oxidase-dependent respiration. The synthesis of PFL and cytochrome bd oxidase was found to be maximal in the lower microaerobic range but not in a delta ArcA mutant, and we conclude that the Arc system is more active with respect to regulation of these two positively regulated operons during microaerobiosis than during anaerobiosis.

Acetyltransferases↗
PubMed1999

Kinetic evidence for the PsaE-dependent transient ternary complex photosystem I/Ferredoxin/Ferredoxin:NADP(+) reductase in a cyanobacterium.

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.

Base Sequence↗
PubMed1999

Kinetics of and intermediates in a photocycle branching reaction of the photoactive yellow protein from Ectothiorhodospira halophila.

We have studied the kinetics of the blue light-induced branching reaction in the photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila, by nanosecond time-resolved UV/Vis spectroscopy. As compared to the parallel dark recovery reaction of the presumed blue-shifted signaling state pB, the light-induced branching reaction showed a 1000-fold higher rate. In addition, a new intermediate was detected in this branching pathway, which, compared to pB, showed a larger extinction coefficient and a blue-shifted absorption maximum. This substantiates the conclusion that isomerization of the chromophore is the rate-controlling step in the thermal photocycle reactions of PYP and implies that absorption of a blue photon leads to cis-->trans isomerization of the 4-hydroxy-cinnamyl chromophore of PYP in its pB state.

Bacterial Proteins↗
PubMed1999

Localization and function of ferredoxin:NADP(+) reductase bound to the phycobilisomes of Synechocystis.

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'.

Bacterial Proteins↗
PubMed1999

Protonation/deprotonation reactions triggered by photoactivation of photoactive yellow protein from Ectothiorhodospira halophila.

Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode. Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein. The latter is observed at very alkaline pH, the former at acidic pH values. At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation. We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation. One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase. Simultaneously, an amino acid side chain(s) (e.g. His-108) changes from a buried to an exposed position. These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle.

Bacterial Proteins↗
PubMed1999

Global conformational changes upon receptor stimulation in photoactive yellow protein.

Biological signal transduction starts with the activation of a receptor protein. Two central questions in signaling are the mechanism of activation by a stimulus and the nature and extent of the protein conformational changes involved. We report extensive evidence for the occurrence of large structural changes upon the light activation of photoactive yellow protein (PYP), a eubacterial photosensor. Absorption of a blue photon by the p-coumaric acid (pCA) chromophore in pG, the initial state of PYP, results in the formation of pB, a putative signaling state. In the presence of an adequate hydration shell, large structural changes in the protein backbone, involving both solvent accessible and core regions, were detected using Fourier transform infrared (FTIR) difference spectroscopy. A significant part (23%) of the amide groups which are buried in pG become exposed to the solvent in pB, as measured through light-induced H/D exchange, using both electrospray ionization mass spectrometry and FTIR spectroscopy. Exposure of previously buried hydrophobic sites would lead to an increase in heat capacity during pB formation and a decrease in heat capacity during pB decay. Thermodynamic studies indeed show that the heat capacity change of pB activation is -2.35 +/- 0.08 kJ/(mol/K), independent of pH from pH 2.4-7.5. A model for photoactivation of PYP is proposed, which provides a framework for a deeper understanding of receptor activation in general.

Bacteria↗
PubMed1999

The genes rubA and rubB for alkane degradation in Acinetobacter sp. strain ADP1 are in an operon with estB, encoding an esterase, and oxyR.

Alkanes are oxidized in Acinetobacter sp. strain ADP1 by a three-component alkane monooxygenase, composed of alkane hydroxylase, rubredoxin, and rubredoxin reductase. rubA and rubB encode rubredoxin and a NAD(P)H-dependent rubredoxin reductase. We demonstrate here that single base pair substitutions in rubA or rubB lead to defects in alkane degradation, showing that both genes are essential for alkane utilization. Differences in the degradation capacity for hexadecane and dodecane in these mutants are discussed. Two genes, estB and oxyR, are located downstream of rubB, but are not necessary for alkane degradation. estB encodes a functional esterase. oxyR encodes a LysR-type transcriptional regulator, conferring resistance to hydrogen peroxide. rubA, rubB, estB, and oxyR constitute an operon, which is constitutively transcribed from a sigma70 promoter, and an estB-oxyR containing message is also transcribed from an internal promoter.

Acinetobacter↗
PubMed1998

Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy.

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.

Benzylidene Compounds↗
PubMed1998

Solution structure and backbone dynamics of the photoactive yellow protein.

The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy. The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides. The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms. Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions. The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure. To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange. Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics. The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.

Bacterial Proteins↗
PubMed1998

Trans/cis (Z/E) photoisomerization of the chromophore of photoactive yellow protein is not a prerequisite for the initiation of the photocycle of this photoreceptor protein.

The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a "locked" chromophore (through reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybrid II). These hybrids absorb maximally at 464 and 443 nm, respectively, which indicates that in both hybrids the deprotonated chromophore does fit into the chromophore-binding pocket. Because the triple bond cannot undergo cis/trans (or E/Z) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle. Surprisingly, both are able. We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-transform infrared (FTIR) spectroscopy. Both hybrids, upon photoexcitation, display authentic photocycle signals in terms of a red-shifted intermediate; hybrid I, in addition, goes through a blue-shifted-like intermediate state, with very slow kinetics. We interpret these results as further evidence that rotation of the carbonyl group of the thioester-linked chromophore of PYP, proposed in a previous FTIR study and visualized in recent time-resolved x-ray diffraction experiments, is of critical importance for photoactivation of PYP.

Bacterial Proteins↗
PubMed1998

Sequence, chromophore extraction and 3-D model of the photoactive yellow protein from Rhodobacter sphaeroides.

The photoactive yellow protein (pyp) gene has been isolated from Rhodobacter sphaeroides by probing with a homologous PCR-product. A sequence analysis shows that this pyp gene encodes a 124 AA protein with 48% identity to the three known PYPs. Downstream from pyp, a number of adjacent open reading frames were identified, including a gene encoding a CoA-ligase homologue (pCL). This latter protein is proposed to be involved in PYP chromophore activation, required for attachment to the apoprotein. We have demonstrated the presence of the chromophoric group, previously identified in PYP from Ectothiorhodospira halophila as trans 4-hydroxy cinnamic acid, in phototrophically cultured R. sphaeroides cells by capillary zone electrophoresis. The basic structure of the chromophore binding pocket in PYP has been conserved, as shown by a 3D model of R. sphaeroides PYP, constructed by homology-based molecular modelling. In addition, this model shows that R. sphaeroides PYP contains a characteristic, positively charged patch.

Amino Acid Sequence↗
PubMed1998

Current topics in signal transduction in bacteria.

Among the signal transfer systems in bacteria two types predominate: two-component regulatory systems and quorum sensing systems. Both types of system can mediate signal transfer across the bacterial cell envelope; however, the signalling molecule typically is not taken up into the cells in the former type of system, whereas it usually is in the latter. The Two-component systems include the recently described (eukaryotic) phosphorelay systems; quorum sensing systems can be based upon autoinducers of the N-acylated homoserine lactones, and on autoinducers of a peptidic nature. A single bacterial cell contains many signalling modules that primarily operate in parallel. This may give rise to neural-network behaviour. Recently, however, for both types of basic signal transfer modules, it has been demonstrated that they also can be organised in series (i.e. in a hierarchical order). Besides their hierarchical position in the signal transduction network of the cell, the spatial distribution of individual signalling modules may also be an important factor in their efficiency in signal transfer. Many challenges lie hidden in future work to understand these signal transfer processes in more detail. These are discussed here, with emphasis on the mutual interactions between different signal transfer processes. Successful contributions to this work will require rigorous mathematical modelling of the performance of signal transduction components, and -networks, as well as studies on light-sensing signal transduction systems, because of the unsurpassed time resolution obtainable in those latter systems, the opportunity to apply repeated reproducible stimuli, etc. The increased understanding of bacterial behaviour that already has resulted--and may further result--from these studies, can be used to fine-tune the beneficial activities of bacteria and/or more efficiently inhibit their deleterious ones.

Bacteria↗
PubMed1998

Characterization and transcriptional regulation of the Synechocystis PCC 6803 petH gene, encoding ferredoxin-NADP+ oxidoreductase: involvement of a novel type of divergent operator.

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.

Bacterial Proteins↗
PubMed1998

Structural and dynamic changes of photoactive yellow protein during its photocycle in solution.

Light irradiation of photoactive yellow protein (PYP) induces a photocycle, in which red-shifted (pR) and blue-shifted (pB) intermediates have been characterized. An NMR study of the long-lived pB intermediate now reveals that it exhibits a large degree of disorder and exists as a family of multiple conformers that exchange on a millisecond time scale. This shows that the behavior of PYP in solution is different from what has been observed in the crystalline state. Furthermore, differential refolding to ground state pG is observed, whereby the central beta-sheet and parts of the helical structure are formed first and the region around the chromophore at a later stage.

Bacterial Proteins↗
PubMed1998

Concerted motions in the photoactive yellow protein.

Molecular dynamics simulations have been performed with the aim of identifying concerted backbone motions in the photoactive yellow protein. Application of the essential dynamics method revealed large, chromophore-linked fluctuations of the protein in the ground state, as well as in a form containing the isomerized chromophore. Various loops become more mobile upon isomerization of the chromophore, including a loop which is part of the PAS domain motif, found in light perception proteins. The hinge points identified in these fluctuations correlate with the positions of evolutionary conserved glycines. The results derived from the simulations directly correlate with available experimental data, provide a framework for understanding the dynamic behaviour of the yellow protein and give clues to subsequent steps in the signal transduction pathway.

Amino Acid Sequence↗
PubMed1997

Uptake and processing of DNA by Acinetobacter calcoaceticus--a review.

In natural transformation, DNA in the form of macromolecular fragments can be translocated across the cell envelope of prokaryotic microorganisms. During the past two decades, several, largely mutually contradictory, hypotheses have been forwarded to explain the molecular mechanism and bioenergetics of this translocation process. Other biomacromolecules are translocated across the bacterial cell envelope as well, such as polysaccharides and proteins, the latter for instance in the process of the assembly of type-IV pili. This brings up the question whether or not common components are involved. Here, we review analyses of DNA translocation in Acinetobacter calcoaceticus, a Gram-negative eubacterium that is able to migrate through twitching motility, and also shows a high frequency of natural transformation. DNA uptake in this organism is an energy-dependent process. Upon entry into the cells, the DNA fragments are integrated into the resident chromosome when a sufficiently large region of mutual homology is available (200 to 400 bp). However, this process is rather inefficient, and on the average 500 bp of each incoming fragment is degraded through exonuclease activity. Upon covalent attachment of a bulky protein molecule to the transforming DNA, the DNA-translocation machinery becomes blocked in further translocation activity. Since A. calcoaceticus is not well suited for transposon mutagenesis, a random mutagenesis procedure has been developed, based on the ligation of an antibiotic-resistance marker to random fragments of chromosomal DNA. This method was used to generate several mutants impaired in the natural transformation process. Three of these have been characterized in detail. No components, common to the translocation of macromolecules through the cell envelope of Acinetobacter, have been detected in this screen.

Acinetobacter calcoaceticus↗
PubMed1996

Spectral tuning, fluorescence, and photoactivity in hybrids of photoactive yellow protein, reconstituted with native or modified chromophores.

Photoactive yellow proteins (PYPs) constitute a new class of eubacterial photoreceptors, containing a deprotonated thiol ester-linked 4-hydroxycinnamic acid chromophore. Interactions with the protein dramatically change the (photo)chemical properties of this cofactor. Here we describe the reconstitution of apoPYP with anhydrides of various chromophore analogues. The resulting hybrid PYPs, their acid-denatured states, and corresponding model compounds were characterized with respect to their absorption spectrum, pK for chromophore deprotonation, fluorescence quantum yield, and Stokes shift. Three factors contributing to the tuning of the absorption of the hybrid PYPs were quantified: (i) thiol ester bond formation, (ii) chromophore deprotonation, and (iii) specific chromophore-protein interactions. Analogues lacking the 4-hydroxy substituent lack both contributions (chromophore deprotonation and specific chromophore-protein interactions), confirming the importance of this substituent in optical tuning of PYP. Hydroxy and methoxy substituents in the 3- and/or 5-position do not disrupt strong interactions with the protein but increase their pK for protonation and the fluorescence quantum yield. Both deprotonation and binding to apoPYP strongly decrease the Stokes shift of chromophore fluorescence. Therefore, coupling of the chromophore to the apoprotein not only reduces the energy gap between its ground and excited state but also the extent of reorganization between these two states. Two of the PYP hybrids show photoactivity comparable with native PYP, although with retarded recovery of the initial state.

Bacterial Proteins↗