Biomedical subjects

Peter Hegemann

Publications and source records attributed to Peter Hegemann.

At least 19 recordsLinked to original sources

PubMed2006

Fast manipulation of cellular cAMP level by light in vivo.

The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of the flavoproteins PACalpha and PACbeta. Here we report functional expression of PACs in Xenopus laevis oocytes, HEK293 cells and in Drosophila melanogaster, where neuronal expression yields light-induced changes in behavior. The activity of PACs is strongly and reversibly enhanced by blue light, providing a powerful tool for light-induced manipulation of cAMP in animal cells.

Adenylyl Cyclases↗

PubMed2006

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor.

BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae, where they control a range of physiological responses including photosynthesis gene expression, photophobia, and negative phototaxis. Other than in well known photoreceptors such as the rhodopsins and phytochromes, BLUF domains are sensitive to light through an oxidized flavin rather than an isomerizable cofactor. To understand the physicochemical basis of BLUF domain photoactivation, we have applied femtosecond transient absorption spectroscopy to the Slr1694 BLUF domain of Synechocystis PCC6803. We show that photoactivation of BLUF domains proceeds by means of a radical-pair mechanism, driven by electron and proton transfer from the protein to the flavin, resulting in the transient formation of anionic and neutral flavin radical species that finally result in the long-lived signaling state on a 100-ps timescale. A pronounced deuteration effect is observed on the lifetimes of the intermediate radical species, indicating that proton movements underlie their molecular transformations. We propose a photoactivation mechanism that involves a successive rupture of hydrogen bonds between a conserved tyrosine and glutamine by light-induced electron transfer from tyrosine to flavin and between the glutamine and flavin by subsequent protonation at flavin N5. These events allow a reorientation of the conserved glutamine, resulting in a switching of the hydrogen-bond network connecting the chromophore to the protein, followed by radical-pair recombination, which locks the glutamine in place. It is suggested that the redox potential of flavin generally defines the light sensitivity of flavin-binding photoreceptors.

Bacterial Proteins↗

PubMed2006

H+ -pumping rhodopsin from the marine alga Acetabularia.

An opsin-encoding cDNA was cloned from the marine alga Acetabularia acetabulum. The cDNA was expressed in Xenopus oocytes into functional Acetabularia rhodopsin (AR) mediating H+ carried outward photocurrents of up to 1.2 microA with an action spectrum maximum at 518 nm (AR518). AR is the first ion-pumping rhodopsin found in a plant organism. Steady-state photocurrents of AR are always positive and rise sigmoidally from negative to positive transmembrane voltages. Numerous kinetic details (amplitudes and time constants), including voltage-dependent recovery of the dark state after light-off, are documented with respect to their sensitivities to light, internal and external pH, and the transmembrane voltage. The results are analyzed by enzyme kinetic formalisms using a simplified version of the known photocycle of bacteriorhodopsin (BR). Blue-light causes a shunt of the photocycle under H+ reuptake from the extracellular side. Similarities and differences of AR with BR are pointed out. This detailed electrophysiological characterization highlights voltage dependencies in catalytic membrane processes of this eukaryotic, H+ -pumping rhodopsin and of microbial-type rhodopsins in general.

Acetabularia↗

PubMed2006

The photochemistry of the light-, oxygen-, and voltage-sensitive domains in the algal blue light receptor phot.

Phot proteins are blue light photoreceptors in plants and algae that mainly regulate photomovement responses. They contain two light-, oxygen-, and voltage-sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN) as chromophore. Upon blue light illumination, the LOV domains undergo a photocycle, transiently forming a covalent adduct of the FMN moiety with a nearby cysteine residue. The presence of two light-sensitive domains in the photoreceptor raises the question about the differences in properties and function between LOV1 and LOV2. As a model system, the photocycles of the LOV1 and LOV2 domains from phot of the green alga Chlamydomonas reinhardtii have been studied in detail, both separately and in a tandem construct. Here we give an overview about the results on the individual behavior of the domains and their interaction. Furthermore, the current status in the understanding of the role of LOV1 in phot in general is presented.

Animals↗

PubMed2005

Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin.

Techniques for fast noninvasive control of neuronal excitability will be of major importance for analyzing and understanding neuronal networks and animal behavior. To develop these tools we demonstrated that two light-activated signaling proteins, vertebrate rat rhodopsin 4 (RO4) and the green algae channelrhodospin 2 (ChR2), could be used to control neuronal excitability and modulate synaptic transmission. Vertebrate rhodopsin couples to the Gi/o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltage-gated Ca2+ channels. Light-mediated activation of RO4 in cultured hippocampal neurons reduces neuronal firing within ms by hyperpolarization of the somato-dendritic membrane and when activated at presynaptic sites modulates synaptic transmission and paired-pulse facilitation. In contrast, somato-dendritic activation of ChR2 depolarizes neurons sufficiently to induce immediate action potentials, which precisely follow the ChR2 activation up to light stimulation frequencies of 20 Hz. To demonstrate that these constructs are useful for regulating network behavior in intact organisms, embryonic chick spinal cords were electroporated with either construct, allowing the frequency of episodes of spontaneous bursting activity, known to be important for motor circuit formation, to be precisely controlled. Thus light-activated vertebrate RO4 and green algae ChR2 allow the antagonistic control of neuronal function within ms to s in a precise, reversible, and noninvasive manner in cultured neurons and intact vertebrate spinal cords.

Animals↗

PubMed2005

Multiple photocycles of channelrhodopsin.

Two rhodopsins with intrinsic ion conductance have been identified recently in Chlamydomonas reinhardtii. They were named "channelrhodopsins" ChR1 and ChR2. Both were expressed in Xenopus laevis oocytes, and their properties were studied qualitatively by two electrode voltage clamp techniques. ChR1 is specific for H+, whereas ChR2 conducts Na+, K+, Ca2+, and guanidinium. ChR2 responds to the onset of light with a peak conductance, followed by a smaller steady-state conductance. Upon a second stimulation, the peak is smaller and recovers to full size faster at high external pH. ChR1 was reported to respond with a steady-state conductance only but is demonstrated here to have a peak conductance at high light intensities too. We analyzed quantitatively the light-induced conductance of ChR1 and developed a reaction scheme that describes the photocurrent kinetics at various light conditions. ChR1 exists in two dark states, D1 and D2, that photoisomerize to the conducting states M1 and M2, respectively. Dark-adapted ChR1 is completely arrested in D1. M1 converts into D1 within milliseconds but, in addition, equilibrates with the second conducting state M2 that decays to the second dark state D2. Thus, light-adapted ChR1 represents a mixture of D1 and D2. D2 thermally reconverts to D1 in minutes, i.e., much slower than any reaction of the two photocycles.

Animals↗

PubMed2005

The phot LOV2 domain and its interaction with LOV1.

Phot proteins are homologs of the blue-light receptor phototropin. We report a comparative study of the photocycles of the isolated, light-sensitive domains LOV1 and LOV2 from Chlamydomonas reinhardtii phot protein, as well as the construct LOV1/2 containing both domains. Transient absorption measurements revealed a short lifetime of the LOV2-wt triplet state (500 ns), but a long lifetime (287 micros) of the triplet in the mutant LOV2-C250S, in which the reactive cysteine is replaced by serine. For LOV1, in comparison, corresponding numbers of 800 ns and 4 micros for the two conformers in LOV1-wt, and 27 micros for LOV1-C57S have been reported. The triplet decay kinetics in the mixed domains LOV1/2-wt, LOV1/2-C57S, and LOV1/2-C250S can be analyzed as the superposition of the behavior of the corresponding single domains. The situation is different for the slow, thermal reaction of the photoadduct back to the dark form. Whereas the individual domains LOV1 and LOV2 show two decay components, the double domains LOV1/2-C57S and LOV1/2-C250S both show only a single component. The interaction of the two domains does therefore not manifest itself during the lifetime of the triplet states, but changes the decay behavior of the adduct states.

Animals↗

PubMed2005

Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage.

The success of long polynucleotide de novo synthesis is largely dependent on the quality and purity of the oligonucleotides used. Generally, the primary product of any synthesis reaction is directly cloned, and clones with correct products have to be identified. In this study, a novel strategy has been established for removing undesired sequence variants from primary gene synthesis products. Single base-pair mismatches, insertions and deletions were cleaved with specific endonucleases. Three different enzymes--T7 endonuclease I, T4 endonuclease VII and Escherichia coli endonuclease V--have been tested. As a model, a synthetic polynucleotide encoding the bacterial chloramphenicol-acetyltransferase (cat) was synthesized using different methods for one step polynucleotide synthesis based on ligation of oligonucleotides. The influence of enzymatic mismatch cleavage (EMC) as an error correction step on the frequency of correct products was analyzed by functional cloning of the synthetic cat and comparing the error rate with that of untreated products. Significant reduction of all mutation types was observed. Statistical analysis revealed that the T4 and E.coli endonucleases reduced the occurrence of mutations in cloned synthetic gene products. The EMC treatment was successful especially in the removal of deletions and insertions from the primary ligation products.

Base Pair Mismatch↗

PubMed2005

Nuclear-gene targeting by using single-stranded DNA avoids illegitimate DNA integration in Chlamydomonas reinhardtii.

Homologous DNA recombination (HR) allows the deletion (knockout), repair (rescuing), and modification of a selected gene, thereby rendering a functional analysis of the gene product possible. However, targeting of nuclear genes has been an inefficient process in most eukaryotes, including algae, plants, and animals, due to the dominance of integration of the applied DNA into nonhomologous regions of the genome. We have shown for the green alga Chlamydomonas reinhardtii by repairing a previously introduced truncated aminoglycoside 3'-phosphotransferase gene, aphVIII, that single-stranded DNA can recombine with a homologous endogenous DNA region of interest. Nonhomologous DNA integration appeared to be more than 100-fold reduced compared with the use of double-stranded DNA, thus allowing isolation of the homologous recombinants. We propose that this method will be applicable to direct targeting of nuclear C. reinhardtii genes.

Animals↗

PubMed2004

On the reaction mechanism of adduct formation in LOV domains of the plant blue-light receptor phototropin.

The blue-light sensitive photoreceptor, phototropin, is a flavoprotein which regulates the phototropism response of higher plants. The photoinduced triplet state and the photoreactivity of the flavin-mononucleotide (FMN) cofactor in two LOV domains of Avena sativa, Adiantum capillus-veneris, and Chlamydomonas reinhardtii phototropin have been studied by time-resolved electron paramagnetic resonance (EPR) and UV-vis spectroscopy at low temperatures (T < or = 80 K). Differences in the electronic structure of the FMN as reflected by altered zero-field splitting parameters of the triplet state could be correlated with changes in the amino acid composition of the binding pocket in wild-type LOV1 and LOV2 as well as in mutant LOV domains. Even at cryogenic temperatures, time-resolved EPR experiments indicate photoreactivity of the wild-type LOV domains, which was further characterized by UV-vis spectroscopy. Wild-type LOV1 and LOV2 were found to form an adduct between the FMN cofactor and the functional cysteine with a yield of 22% and 68%, respectively. The absorption maximum of the low-temperature photoproduct of wild-type LOV2 is red-shifted by about 15 nm as compared with the FMN C(4a)-cysteinyl adduct formed at room temperature. In light of these observations, we discuss a radical-pair reaction mechanism for the primary photoreaction in LOV domains.

Adiantum↗

PubMed2004

Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene.

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii . This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii .

Algal Proteins↗

PubMed2004

Recording of blue light-induced energy and volume changes within the wild-type and mutated phot-LOV1 domain from Chlamydomonas reinhardtii.

The time-resolved thermodynamics of the flavin mononucleotide (FMN)-binding LOV1 domain of Chlamydomonas reinhardtii phot (phototropin homolog) was studied by means of laser-induced optoacoustic spectroscopy. In the wild-type protein the early red-shifted intermediate LOV(715) exhibits a small volume contraction, DeltaV(715) = -1.50 ml/mol, with respect to the parent state. LOV(715) decays within few micro s into the covalent FMN-Cys-57 adduct LOV(390), that shows a larger contraction, DeltaV(390) = -8.8 ml/mol, suggesting a loss of entropy and conformational flexibility. The high energy content of LOV(390), E(390) = 180 kJ/mol, ensures the driving force for the completion of the photocycle and points to a strained photoreceptor conformation. In the LOV-C57S mutated protein the photoadduct is not formed and DeltaV(390) is undetected. Large effects on the measured DeltaVs are observed in the photochemically competent R58K and R58K/D31Q mutated proteins, with DeltaV(390) = -2.0 and -1.9 ml/mol, respectively, and DeltaV(715) approximately 0. The D31Q and D31N substitutions exhibit smaller but well-detectable effects. These results show that the photo-induced volume changes involve the protein region comprising Arg-58, which tightly interacts with the FMN phosphate group.

Amino Acid Substitution↗

PubMed2004

"Vision" in single-celled algae.

Photosynthetic unicellular algae have a unique visual system. In Chlamydomonas reinhardtii, the pigmented eye comprises the optical system and at least five different rhodopsin photoreceptors. Two of them, the channelrhodopsins, are rhodopsin-ion channel hybrids switched between closed and open states by photoisomerization of the attached retinal chromophore. They promise to become a useful tool for noninvasive control of membrane potential and intracellular ion concentrations.

Animals↗

PubMed2003

Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.

Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane alpha helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination.

Algal Proteins↗

PubMed2003

Irreversible photoreduction of flavin in a mutated Phot-LOV1 domain.

Phot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN). Upon absorption of blue light, the LOV domains undergo a photocycle, transiently forming a covalent adduct of a cysteine residue and the FMN (LOV-390). The mechanism of formation of this flavin-thiol adduct is still unclear. We studied a mutant of the LOV1 domain from the green alga Chlamydomonas reinhardtii with a methionine replacing the reactive cysteine 57 (C57M). As in the wild type, irradiation leads to formation of a photoadduct, which, however, is irreversibly converted into a red absorbing species, C57M-675. On the basis of spectroscopic results and the 2.1 A resolution crystal structure, this highly unusual FMN species was assigned to a neutral flavin radical covalently attached to the apoprotein at the N(5) position. In contrast to other flavoprotein neutral radicals, C57M-675 is stable even under aerobic or denaturing conditions. Pathways for the photoinduced formation of the adduct are discussed for the C57M mutant as well as the wild-type LOV1 domain.

Animals↗

PubMed2003

Characterization of a flavin radical product in a C57M mutant of a LOV1 domain by electron paramagnetic resonance.

In the flavin mononucleotide-binding LOV1 domain of the Phot1-receptor from Chlamydomonas reinhardtii the photoreactive cysteine C57 has been replaced by methionine. Photoexcitation of this C57M mutant yields a metastable photoproduct (C57M-415) that thermally decomposes into a stable paramagnetic species (C57M-675) with extremely red-shifted absorption in the visible range. In this contribution, we describe the characterization of this radical by multi-frequency electron paramagnetic resonance and electron-nuclear double resonance. The main features of the spectra identify the paramagnetic species as a flavin neutral radical. However, detailed analysis shows that the isoalloxazine moiety of the flavin is alkyl substituted at N(5), rather than protonated as is usually the case. The implication of these observations on the likely mechanism of photoproduct generation in wild-type LOV domains is discussed.

Amino Acid Substitution↗

PubMed2003

Phot-LOV1: photocycle of a blue-light receptor domain from the green alga Chlamydomonas reinhardtii.

The "Phot" protein family comprises blue-light photoreceptors that consist of two flavin mononucleotide (FMN)-binding LOV (light, oxygen, and voltage) domains and a serine/threonine kinase domain. We have investigated the LOV1 domain of Phot1 from Chlamydomonas reinhardtii by time-resolved absorption spectroscopy. Photoexcitation of the dark form, LOV1-447, causes transient bleaching and formation of two spectrally similar red-shifted intermediates that are both assigned to triplet states of the FMN. The triplet states decay with time constants of 800 ns and 4 micro s with an efficiency of >90% into a blue-shifted intermediate, LOV1-390, that is attributed to a thiol adduct of cysteine 57 to FMN C(4a). LOV1-390 reverts to the dark form in hundreds of seconds, the time constant being dependent on pH and salt concentration. In the mutant C57S, where the thiol adduct cannot be formed, the triplet state displays an oxygen-dependent decay directly to the dark form. We present here a spectroscopic characterization of an algal sensory photoreceptor in general and of a LOV1 domain photocycle in particular. The results are discussed with respect to the behavior of the homologous LOV2 domain from oat.

Animals↗

PubMed2003

Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii.

Phot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptors consist of two flavin mononucleotide (FMN)-binding light, oxygen, or voltage (LOV) domains and a C-terminal serine/threonine kinase domain. We determined crystal structures of the LOV1 domain of Phot1 from the green alga Chlamydomonas reinhardtii in the dark and illuminated state to 1.9 A and 2.8 A resolution, respectively. The structure resembles that of LOV2 from Adiantum (Crosson, S. and K. Moffat. 2001. PROC: Natl. Acad. Sci. USA. 98:2995-3000). In the resting dark state of LOV1, the reactive Cys-57 is present in two conformations. Blue-light absorption causes formation of a proposed active signaling state that is characterized by a covalent bond between the flavin C4a and the thiol of Cys-57. There are differences around the FMN chromophore but no large overall conformational changes. Quantum chemical calculations based on the crystal structures revealed the electronic distribution in the active site during the photocycle. The results suggest trajectories for electrons, protons, and the active site cysteine and offer an interpretation of the reaction mechanism.

Amino Acid Sequence↗