Hugo Scheer - Profile on Academia.edu (original) (raw)

Papers by Hugo Scheer

Advances in Photosynthesis Research, 1984

The Journal of Chemical Physics, 1994

Zero field absorption detected magnetic resonance hole burning measurements were performed on pho... more Zero field absorption detected magnetic resonance hole burning measurements were performed on photosynthetic reaction centers of the bacteria Rhodobacter sphaeroides R26 and Rhodopseudomonas viridis. Extrapolation to zero microwave power yielded pseudohomogeneous linewidths of 2.0 MHz for Rhodopseudomonas viridis, 1.0 and 0.9 MHz for the protonated forms of Rhodobacter sphaeroides R26 with and without monomer bacteriochlorophyll exchanged, and 0.25 MHz as an upper limit for fully deuterated reaction centers of Rhodobacter sphaeroides R26. The measured linewidths were interpreted as being due to unresolved hyperfine interaction between the nuclear spins and the triplet electron spin, the line shape being determined by spectral diffusion among the nuclei. The difference in linewidths between Rhodobacter sphaeroides R26 and Rhodopseudomonas viridis is then explained by triplet delocalization on the special pair in the former, and localization on one dimer half on the latter. In the fully deuterated sample, four quadrupole satellites were observed in the hole spectra arising from the eight 14N nitrogens in the special pair. The quadrupole parameters seem to be very similar for all nitrogens and were determined to ~=1.25tO.l MHz and 7=0.9tO.l MHz.

Photochemistry and Photobiology, 1988

Reaction centers from Rhodobacter sphaeroides have been modified by treatment with sodium borohyd... more Reaction centers from Rhodobacter sphaeroides have been modified by treatment with sodium borohydride similar to the original procedure [Ditson el al., Biochim. Biophys. Acta 766, 623 (1984)], and investigated spectroscopically and by gel electrophoresis. (1) Low temperature (1.2 K) absorption, fluorescence, absorption-and fluorescence-detected ODMR, and microwave-induced singlet-triplet absorption difference spectra (MIA) suggest that the treatment produces a spectroscopically homogeneous preparation with one of the 'additional' bacteriochlorophylls being removed. The modification does not alter the zero field splitting parameters of the primary donor triplet (TP870). (2) From the circular dichroism and Raman resonance spectra in the 1500-1800 c m-' region, the removed pigment is assigned to Bchl,, e.g. the "extra" Bchl on the "inactive" M-branch. (3) A strong coupling among all pigment molecules is deduced from the circular dichroism spectra, because pronounced band-shifts and/or intensity changes occur in the spectral components assigned to all pigments. This is supported by distinct differences among the MIA spectra of untreated and modified reaction centers, as well as by Raman resonance. (4) The modification is accompanied by partial proteolytic cleavage of the M-subunit. The preparation is thus spectroscopically homogeneous, hut biochemically heterogenous.

Procedia Chemistry, 2015

The brightly fluorescent phycobiliproteins are light-harvesting pigments in cyanobacteria and cer... more The brightly fluorescent phycobiliproteins are light-harvesting pigments in cyanobacteria and certain algae, covering almost the entire visible spectrum. Another type of biliproteins, the phytochrome family, comprises regulatory photoswitches in plants, fungi and many bacteria; their spectra cover an even wider range extending into the near-infrared and near-ultraviolet. In both types, the chromophores can be tuned by modulating chromophore-protein interactions, including the transition between fluorescence and photoswitching. These properties render biliproteins useful for bioimaging. Applications require not only the introduction of genes coding for apoproteins, but also genes coding for biosynthesis of the chromophores. Strategies for tuning and heterologous biosynthesis will be discussed.

Significance of threshold doses for photodynamic therapy of melanotic and amelanotic tumors

5th International Photodynamic Association Biennial Meeting, 1994

ABSTRACT The decay of the surviving cell fraction, F, during photodynamic treatment of a cell pop... more ABSTRACT The decay of the surviving cell fraction, F, during photodynamic treatment of a cell population, F0, vs. energy dose obeys an exponential law. The integrated logarithmic form yields a straight line with intercept 0 and slope -k. The reciprocal slope yields LD90, i.e., the energy fluence (J/cm2) necessary to kill 90% of the cell population F0. In reality there are deviations from this simple set of equations. At low power densities most photosensitizers are unable to kill cells. This leads to an `optical threshold' without cell destruction but frequently with cell stimulating effects, so called `overshots.' This general phenomenon occurs in a very pronounced form with melanotic melanoma cells.

Relative binding affinities of chlorophylls in peridinin–chlorophyll–protein reconstituted with heterochlorophyllous mixtures

Photosynthesis Research, 2007

Peridinin-chlorophyll-protein (PCP), containing differently absorbing chlorophyll derivatives, ar... more Peridinin-chlorophyll-protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these "half-mers", homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.

Biochemistry, 2001

Replacement of the central Mg in chlorophylls by Ni opens an ultrafast (tens of femtoseconds time... more Replacement of the central Mg in chlorophylls by Ni opens an ultrafast (tens of femtoseconds time range) radiationless de-excitation path, while the principal ground-state absorption and coordination properties of the pigment are retained. A method has been developed for substituting the native bacteriochlorophyll a by Ni-bacteriochlorophyll a ([Ni]-BChl) in the light harvesting antenna of the core complex (LH1) from the purple bacterium, Rhodobacter (Rb.) sphaeroides, to investigate its unit size and excited state properties. The components of the complex have been extracted with an organic solvent from freeze-dried membranes of an LH1-only strain of Rb. sphaeroides and transferred into the micelles of n-octyl-glucopyranoside (OG). Reconstitution was achieved by solubilization in 3.4% OG, followed by dilution, yielding a complex nearly identical to the native one, in terms of absorption, fluorescence, and circular dichroism spectra as well as energy transfer efficiency from carotenoid to bacteriochlorophyll. By adding increasing amounts of [Ni]-BChl to the reconstitution mixture, a series of LH1 complexes was obtained that contain increasing levels of this efficient excitation trap. In contrast to the nearly unchanged absorption, the presence of [Ni]-BChl in LH1 markedly affects the emission properties. Incorporation of only 3.2 and 20% [Ni]-BChl reduces the emission by 50% and nearly 100%, respectively. The subnanosecond fluorescence kinetics of the complexes were monoexponential, with the lifetime identical to that of the native complex, and its amplitude decreasing in parallel with the steady-state fluorescence yield. Quantitative analysis of the data, based on a Poisson distribution of the modified pigment in the reconstituted complex, suggests that the presence of a single excitation trap per LH1 unit suffices for efficient emission quenching and that this unit contains 20 (1 BChl molecules. † The project was supported by the Deutsche Forschungsgemeinschaft (Sche 140/9-3 and SFB 533). L.F. acknowledges the postdoctoral fellowship provided by the Alexander von Humboldt Foundation.

FEBS Letters, 2002

Native and carotenoid-depleted peripheral purple bacterial light-harvesting complex (LH2) were in... more Native and carotenoid-depleted peripheral purple bacterial light-harvesting complex (LH2) were investigated by simultaneous two-photon excited (between 1300^1500 nm) £uorescence (TPF). TPF results from direct bacteriochlorophyll excitation in both samples. The spectral position of the 2A 3 g state of rhodopsin is indicated by a diminuition of the bacteriochlorophyll TPF in native LH2. In conclusion, comparison to carotenoid-depleted samples is a conditio sine qua non for unambiguous interpretation of similar experiments.

Springer Series in Biophysics, 1990

Towards a more accurate future for chlorophyll a and b determinations: the inaccuracies of Daniel Arnon’s assay

Photosynthesis Research

A recent publication (Esteban in New Phytol 217:341–342, 2018) describes how the use and citation... more A recent publication (Esteban in New Phytol 217:341–342, 2018) describes how the use and citation of the assay of chlorophylls a and b extracted in aqueous 80% acetone by Arnon (Plant Physiol 2:1–15, 1949) is increasing, even in journals with high impact factors. This is a very disconcerting situation: the assay is outdated because it relies on the seriously under-estimated extinction coefficients of Mackinney (J Biol Chem 140:315–322, 1941), and the assay of chlorophylls is one of the most important, and much reported, procedures in studies of photosynthesis and related plant biological fields. Using the assay has led to the accumulation of masses of inaccurate data and confusion during the resolution of some plant biological problems. A summary not only of an accurate assay of chlorophylls in aqueous 80% acetone but also of a long-known method to correct the data obtained by Arnon’s procedure (cf. Porra et al. in Biochim Biophys Acta 975:384–394, 1989) is briefly described below together with references to reliable assays in this and other solvents by other authors.

Far-red acclimating cyanobacterium as versatile source for bright fluorescent biomarkers

Biochimica et biophysica acta. Molecular cell research, 2018

Far-red and near-infrared emitting chromophores extend applications of fluorescent proteins to re... more Far-red and near-infrared emitting chromophores extend applications of fluorescent proteins to regions of maximal transmission of most tissues, but present considerable engineering challenges. Far-red adapting cyanobacteria generate a novel set of biliproteins. One of them, ApcF2, from a thermophilic cyanobacterium was subjected to structure-guided, site-directed random and specific mutagenesis, and was screened for bright far-red emission. We report the generation of chromoproteins, termed BDFPs, that are small, bind auto-catalytically the ubiquitous biliverdin as chromophore, express well, and retain their fluorescence in mammalian cells and in the nematode, C. elegans. They are, moreover, photostable and tolerate high temperature, low pH and chemical denaturation. Homo-bichromophoric tandems of these proteins improve labeling, while hetero-bichromophoric systems with large Stokes shifts are suitable for applications like FRET, multi-channel or super-resolution microscopy. The BDF...

Tetrahedron

Die diastereomeren C-9-Alkohole 3% b-l, 2 und da, b-1, 2 wurden durch NaBH,-Reduktion der I@AIkox... more Die diastereomeren C-9-Alkohole 3% b-l, 2 und da, b-1, 2 wurden durch NaBH,-Reduktion der I@AIkoxy-phlophorbide la, b und 2a, b dargestellt. Ihre absolute Konfiguration an C-10 wurde durch NMR-und ORD/CD-Messungen sowie durch chemische Korrelation bestimmt, die Konfiguration an C-9 durch IR-und vor allem NMR-Spektroskopie. Hierzu muuten mit Hilfe da selektiv deuterierten C-9-Alkohole 5a, b-l, 2 die NMR-Spektren von 38, b-l, 2 vollstandig zugeordnet werden. Unter den alkalis&en Reduktionsbedingunge ist die C-IO-Kontiguration stabiI wahrend am C-9 teilweise Epimerisierung erfolgt. Die saure Alkoholyse von 3a, b-1, 2 verlluft dagegen unter iiquilibrierung an C-IO ti weitgehender Retention an C-9. Die Wasserstoflbriicken zwischen den C-IO-Substituenten (-CGGCH,,-OCHs) und der 9-OH-Gruppe wurden NMR-und IR-spektroskopisch untersucht .

Small monomeric and highly stable near-infrared fluorescent markers derived from the thermophilic phycobiliprotein, ApcF2

Biochimica et biophysica acta, Oct 1, 2017

Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal ... more Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing, but their application presents considerable challenges. Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases. In addition, their fluorescence yield generally decreases with increasing wavelengths and depends strongly on the state of the binding protein. We report fluorescent biliproteins, termed BDFPs, that are derived from the phycobilisome core subunit, ApcF2: this subunit is induced in the thermophilic cyanobacterium, Chroococcidiopsis thermalis, by far-red light and binds phycocyanobilin non-covalently. The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region (~710nm). They are small monomers (~15kDa) and...

Chromophorylation (in Escherichia coli) of allophycocyanin B subunits from far-red light acclimated Chroococcidiopsis thermalis sp. PCC7203

Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2017

Cyanobacterial phycobilisomes funnel the harvested light energy to the reaction centers via two t... more Cyanobacterial phycobilisomes funnel the harvested light energy to the reaction centers via two terminal emitters, allophycocyanin B and the core-membrane linker. ApcD is the α-subunit of allophycocyanin B responsible for its red-shifted absorbance (λmax 665 nm). Far-red photo-acclimated cyanobacteria contain certain allophycocyanins that show even further red-shifted absorbances (λmax > 700 nm). We studied the chromophorylation of the three far-red induced ApcD subunits ApcD2, ApcD3 and ApcD4 from Chroococcidiopsis thermalis sp. PCC7203 during the expression in E. coli. The complex behavior emphasizes that a variety of factors contribute to the spectral red-shift. Only ApcD2 bound phycocyanobilin covalently at the canonical position C81, while ApcD3 and ApcD4 gave only traces of stable products. The product of ApcD2 was, however, heterogeneous. The major fraction had a broad absorption around 560 nm and double-peaked fluorescence at 615 and 670 nm. A minor fraction was similar t...

A Simple Preparation Method for Phytochromobilin

Photochemistry and photobiology, May 1, 2017

Phytochromobilin (PΦB), the chromophore of plant phytochromes, is difficult to isolate because ph... more Phytochromobilin (PΦB), the chromophore of plant phytochromes, is difficult to isolate because phytochromes occur at very low concentrations in plants. It is, therefore, frequently replaced in plant phytochrome studies by phycocyanobilin, which is abundant in cyanobacteria. PΦB is also an attractive chromophore for far-red emitting chromoproteins. In this work, we design and optimize a simple method to efficiently isolate useful quantities of PΦB: The chromophore is generated in Escherichia coli and transiently bound to a tailored chromophore-binding domain of ApcE2, the apo-protein of a core-membrane linker, from which it can subsequently be released. The ease and effectiveness of this method hinges not only on the enhanced biosynthesis of PΦB in the presence of the ApcE2 construct from Synechococcus sp. PCC7335, but also on the noncovalent binding of the pigment to its apo-protein. The isolated PΦB was successfully incorporated into phytochrome-related assemblies, and furthermore,...

Carrier systems in PDT: on the way to novel antitumor drugs

Proceedings of SPIE, 1995

A novel mode to apply photosensitizing drugs specifically to tumor tissue using the principles of... more A novel mode to apply photosensitizing drugs specifically to tumor tissue using the principles of polyphasic tumor therapies is outlined. Key compounds are tumor specific functionalized antibodies with reduced immunogenicity. These bind to drug inclusion complexes in a multiplicative manner. Drug inclusion complexes are designed on the basis of tethered functionalized (beta) -cyclodextrin dimers with maximum affinity to porphyrinoid photosensitizers forming monomeric chlathrates. To enhance porphyrin-cyclodextrin interaction peripheral groups of the porphyrin have to be chemically modified. The development of the method is not yet completed. First results are demonstrated.

Phytochrome chromophore and models

Far-red light photoacclimation: Chromophorylation of FR induced α- and β-subunits of allophycocyanin from Chroococcidiopsis thermalis sp. PCC7203

Biochimica et biophysica acta, Sep 29, 2016

Cyanobacterial light-harvesting complexes, phycobilisomes, can undergo extensive remodeling under... more Cyanobacterial light-harvesting complexes, phycobilisomes, can undergo extensive remodeling under varying light conditions. Acclimation to far-red light involves not only generation of red-shifted chlorophylls in the photosystems, but also induction of additional copies of core biliproteins that have been related to red-shifted components of the phycobilisome (Gan et al., Life 5, 4, 2015). We are studying the molecular basis for these acclimations in Chroococcidiopsis thermalis sp. PCC7203. Five far-red induced allophycocyanin subunits (ApcA2, ApcA3, ApcB2, ApcB3 and ApcF2) were expressed in Escherichia coli, together with S-type chromophore-protein lyases and in situ generated chromophore, phycocyanobilin. Only one subunit, ApcF2, shows an unusual red-shift (λAmax~675nm, λFmax~698nm): it binds the chromophore non-covalently, thereby preserving its full conjugation length. This mechanism operates also in two Cys-variants of the induced subunits of bulky APC. All other wild-type subu...