Herstellung und spektroskopische Charakterisierung von Cryptochromen aus Algen zur Aufklärung ihrer Funktionsweise als Photorezeptoren

Spexard M. Herstellung und spektroskopische Charakterisierung von Cryptochromen aus Algen zur Aufklärung ihrer Funktionsweise als Photorezeptoren. Bielefeld; 2012

Infrared spectrum and absorption coefficient of the cofactor flavin in water

Spexard M, Immeln D, Thöing C, Kottke T. Infrared spectrum and absorption coefficient of the cofactor flavin in water. VIBRATIONAL SPECTROSCOPY. 2011;57(2):282-287

Response of the Sensory Animal-like Cryptochrome aCRY to Blue and Red Light As Revealed by Infrared Difference Spectroscopy

Spexard M, Thöing C, Beel B, Mittag M, Kottke T. Response of the Sensory Animal-like Cryptochrome aCRY to Blue and Red Light As Revealed by Infrared Difference Spectroscopy. Biochemistry. 2014;53(6):1041-1050

A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of light-harvesting protein levels

Juhas M, von Zadow A, Spexard M, Schmidt M, Kottke T, Büchel C. A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of light-harvesting protein levels. FEBS Journal. 2014;281(9):2299-2311.Diatoms possess several genes for proteins of the cryptochrome/photolyase family. A typical sequence for a plant cryptochrome was not found in our analysis of the Phaeodactylumtricornutum genome, but one protein grouped with higher plant and green algal cryptochromes. This protein, CryP, binds FAD and 5,10-methenyltetrahydrofolate, according to our spectroscopic studies on heterologously expressed protein. 5,10-Methenyltetrahydrofolate binding is a feature common to both cyclobutane pyrimidine dimer photolyases and DASH cryptochromes. In recombinant CryP, however, the FAD chromophore was present in its neutral radical state and had a red-shifted absorption maximum at 637nm, which is more characteristic for a DASH cryptochrome than a cyclobutane pyrimidine dimer photolyase. Upon illumination with blue light, the fully reduced state of FAD was formed in the presence of reductant. Expression of CryP was silenced by antisense approaches, and the resulting cell lines showed increased levels of proteins of light-harvesting complexes, the Lhcf proteins, invivo. In contrast, the levels of proteins active in light protection, the Lhcx proteins, were reduced. Thus, CryP cannot be directly grouped with known members of the cryptochrome/photolyase family. Of all P.tricornutum proteins, it is the most similar in sequence to a plant cryptochrome, and is involved in the regulation of light-harvesting protein expression, but shows spectroscopic features and a chromophore composition that are most typical of a DASH cryptochrome

Response of the Sensory Animal-like Cryptochrome aCRY to Blue and Red Light As Revealed by Infrared Difference Spectroscopy

Cryptochromes act as blue light sensors in plants, insects, fungi, and bacteria. Recently, an animal-like cryptochrome (aCRY) was identified in the green alga <i>Chlamydomonas reinhardtii</i> by which gene expression is altered in response to not only blue light but also yellow and red light. This unique response of a flavoprotein <i>in vivo</i> has been attributed to the fact that the neutral radical of the flavin chromophore acts as dark form of the sensor, which absorbs in almost the entire visible spectral range (<680 nm). Here, we investigated light-induced processes in the protein moiety of full-length aCRY by UV–vis and Fourier transform infrared spectroscopy. Findings are compared to published results on the homologous (6-4) photolyases, DNA repair enzymes. The oxidized state of aCRY is converted to the neutral radical by blue light. The recovery is strongly dependent on pH and might be catalyzed by a conserved histidine of the (6-4)/clock cluster. The decay is independent of oxygen concentration in contrast to that of other cryptochromes and (6-4) photolyases. This blue light reaction of the oxidized flavin is not accompanied by any detectable changes in secondary structure, in agreement with a role <i>in vivo</i> of an unphysiological preactivation. In contrast, the conversion by red light of the neutral radical to the anionic fully reduced state proceeds with conformational changes in turn elements, which most probably constitute a part of the signaling process. These changes have not been detected in the corresponding transition of (6-4) photolyase, which points to a decisive difference between the sensor and the enzyme

A Flavin-Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii

Beel B, Prager K, Spexard M, et al. A Flavin-Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii. THE PLANT CELL. 2012;24(7):2992-3008.Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue- and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light-activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities

Screening for conditions of enhanced production of a recombinant beta-glucanase secreted into the medium by Escherichia coli

Spexard M, Beshay U, Risse JM, Miksch G, Flaschel E. Screening for conditions of enhanced production of a recombinant beta-glucanase secreted into the medium by Escherichia coli. BIOTECHNOLOGY LETTERS. 2010;32(2):243-248.The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (> 200 U ml(-1)) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol