Ice nucleation and growth is an important and widespread environmental process. Accordingly, nature has developed means to either promote or inhibit ice crystal formation, for example ice-nucleating ...proteins in bacteria or ice-binding antifreeze proteins in polar fish. Recently, it was found that birch pollen release ice-nucleating macromolecules when suspended in water. Here we show that birch pollen washing water exhibits also ice-binding properties such as ice shaping and ice recrystallization inhibition, similar to antifreeze proteins. We present spectroscopic evidence that both the ice-nucleating as well as the ice-binding molecules are polysaccharides bearing carboxylate groups. The spectra suggest that both polysaccharides consist of very similar chemical moieties, but centrifugal filtration indicates differences in molecular size: ice nucleation occurs only in the supernatant of a 100 kDa filter, while ice shaping is strongly enhanced in the filtrate. This finding may suggest that the larger ice-nucleating polysaccharides consist of clusters of the smaller ice-binding polysaccharides, or that the latter are fragments of the ice-nucleating polysaccharides. Finally, similar polysaccharides released from pine and alder pollen also display both ice-nucleating as well as ice-binding ability, suggesting a common mechanism of interaction with ice among several boreal pollen with implications for atmospheric processes and antifreeze protection.
BLUF (blue light sensor using flavin) domains regulate the activity of various enzymatic effector domains in bacteria and euglenids. BLUF features a unique photoactivation through restructuring of ...the hydrogen-bonding network as opposed to a redox reaction or an isomerization of the chromophore. A conserved glutamine residue close to the flavin chromophore plays a central role in the light response, but the underlying modification is still unclear. We labelled this glutamine with (15)N in two representative BLUF domains and performed time-resolved infrared double difference spectroscopy. The assignment of the signals was conducted by extensive quantum chemical calculations on large models with 187 atoms reproducing the UV-vis and infrared signatures of BLUF photoactivation. In the dark state, the comparatively low frequency of 1,667 cm(-1) is assigned to the glutamine C=O accepting a hydrogen bond from tyrosine. In the light state, the signature of a tautomerised glutamine was extracted with the C=N stretch at ~1,691 cm(-1) exhibiting the characteristic strong downshift by (15)N labelling. Moreover, an indirect isotope effect on the flavin C4=O stretch was found. We conclude that photoactivation of the BLUF receptor does not only involve a rearrangement of hydrogen bonds but includes a change in covalent bonds of the protein.
In plants and algae, light serves both as the energy source for photosynthesis and a biological signal that triggers cellular responses via specific sensory photoreceptors. Red light is perceived by ...bilin-containing phytochromes and blue light by the flavin-containing cryptochromes and/or phototropins (PHOTs), the latter containing two photosensory light, oxygen, or voltage (LOV) domains. Photoperception spans several orders of light intensity, ranging from far below the threshold for photosynthesis to values beyond the capacity of photosynthetic CO
assimilation. Excess light may cause oxidative damage and cell death, processes prevented by enhanced thermal dissipation via high-energy quenching (qE), a key photoprotective response. Here we show the existence of a molecular link between photoreception, photosynthesis, and photoprotection in the green alga Chlamydomonas reinhardtii. We show that PHOT controls qE by inducing the expression of the qE effector protein LHCSR3 (light-harvesting complex stress-related protein 3) in high light intensities. This control requires blue-light perception by LOV domains on PHOT, LHCSR3 induction through PHOT kinase, and light dissipation in photosystem II via LHCSR3. Mutants deficient in the PHOT gene display severely reduced fitness under excessive light conditions, indicating that the sensing, utilization, and dissipation of light is a concerted process that plays a vital role in microalgal acclimation to environments of variable light intensities.
Plant cryptochromes regulate the circadian rhythm, flowering time, and photomorphogenesis in higher plants as responses to blue light. In the dark, these photoreceptors bind oxidized FAD in the ...photolyase homology region (PHR). Upon blue light absorption, FAD is converted to the neutral radical state, the likely signaling state, by electron transfer via a conserved tryptophan triad and proton transfer from a nearby aspartic acid. Here we demonstrate, by infrared and time-resolved UV-visible spectroscopy on the PHR domain, that replacement of the aspartic acid Asp-396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by 6 orders of magnitude. This short lifetime does not permit to drive conformational changes in the C-terminal extension that have been associated with signal transduction. Only in the presence of ATP do both the wild type and mutant form a long-lived radical state. However, in the mutant, an anion radical is formed instead of the neutral radical, as found previously in animal type I cryptochromes. Infrared spectroscopic experiments demonstrate that the light-induced conformational changes of the PHR domain are conserved in the mutant despite the lack of proton transfer. These changes are not detected in the photoreduction of the non-photosensory d-amino acid oxidase to the anion radical. In conclusion, formation of the anion radical is sufficient to generate a protein response in plant cryptochromes. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP.Plant cryptochromes are blue light sensors forming an exceptionally stable flavin neutral radical as a signaling state.
Blockage of proton transfer to flavin severely reduces the lifetime of the radical.
The proton donor aspartic acid acts as an intrinsic stabilizer of the signaling state.
The role of a key structural element is identified, distinguishing plant cryptochromes from other members of the family.
Green algae have a highly complex and diverse set of cryptochrome photoreceptor candidates including members of the following subfamilies: plant, plant-like, animal-like, DASH and cryptochrome ...photolyase family 1 (CPF1). While some green algae encode most or all of them, others lack certain members. Here we present an overview about functional analyses of so far investigated cryptochrome photoreceptors from the green algae Chlamydomonas reinhardtii (plant and animal-like cryptochromes) and Ostreococcus tauri (CPF1) with regard to their biological significance and spectroscopic properties. Cryptochromes of both algae have been demonstrated recently to be involved to various extents in circadian clock regulation and in Chlamydomonas additionally in life cycle control. Moreover, CPF1 even performs light-driven DNA repair. The plant cryptochrome and CPF1 are UVA/blue light receptors, whereas the animal-like cryptochrome responds to almost the whole visible spectrum including red light. Accordingly, plant cryptochrome, animal-like cryptochrome and CPF1 differ fundamentally in their structural response to light as revealed by their visible and infrared spectroscopic signatures, and in the role of the flavin neutral radical acting as dark form or signaling state.
Light is important for algae, as it warrants metabolic independence via photosynthesis. In addition to the absorption of light by the photosystems, algae possess a variety of specific photoreceptors ...that allow the quantification of the light fluxes as well as the assessment of light qualities. About a decade ago, aureochromes have been described in the xanthophyte alga Vaucheria frigida. These proteins represent a new type of blue light photoreceptor as they possess both a light-oxygen-voltage (LOV) domain for light reception as well as a basic region leucine zipper (bZIP) domain for DNA binding, indicating that they represent light-driven transcription factors. Aureochromes so far have been detected only in a single group of algae, photosynthetic stramenopiles, but not in any other prokaryotic or eukaryotic organisms. Recent biophysical work on aureochromes in the absence and the presence of DNA revealed the mechanism of allosteric communication between the sensor and effector domains despite their unusual inversed arrangement. Different molecular models have been proposed to describe the effect of light on DNA binding. Functional characterization of mutants of the diatom Phaeodactylum tricornutum, in which the aureochrome genes have been silenced or deleted, indicate that different aureochromes may have different functions, being involved in central processes like light acclimation and regulation of the cell cycle.
Plant cryptochromes are central blue light receptors for the control of land plant and algal development including the circadian clock and the cell cycle. Cryptochromes share a photolyase homology ...region with about 500 amino acids and bind the chromophore flavin adenine dinucleotide. Characteristic for plant cryptochromes is a conserved aspartic acid close to flavin and an exceptionally long C-terminal extension. The mechanism of activation by excitation and reduction of the chromophore flavin adenine dinucleotide has been controversially discussed for many years. Various spectroscopic techniques have contributed to our understanding of plant cryptochromes by providing high time resolution, ambient conditions and even in-cell approaches. As a result, unifying and differing aspects of photoreaction and signal propagation have been revealed in comparison to members from other cryptochrome subfamilies. Here, we review the insight from spectroscopy on the flavin photoreaction in plant cryptochromes and present the current models on the signal propagation from flavin reduction to dissociation of the C-terminal extension.
Plant cryptochromes are light receptors in land plants and algae with very diverse functions such as circadian timing and lifecycle progression. The receptor consists of a photolyase homology region ...(PHR) binding the flavin chromophore and a C-terminal extension (CCT) responsible for signaling. The reputed signaling state, the flavin neutral radical, is formed by a femtosecond electron transfer and microsecond proton transfer to the excited, oxidized flavin. Subsequently, a 500 μs loss of β-sheet structure ∼25 Å away from flavin was resolved and suggested to be part of the signal conduction to the CCT. Here, we performed time-resolved, step-scan Fourier transform IR spectroscopy on the PHR of the plant cryptochrome pCRY (formerly CPH1) from Chlamydomonas reinhardtii. In a mutant lacking the proton donor aspartic acid 396 only the flavin anion radical is formed, but we observed the loss of β-sheet structure with a time constant of 1.3 ms, similar to the 500 μs of the wild type. This finding implies that the anion radical may be considered signaling-competent. In the steady state, a variation of external pH up to 8.3 did not have any effect on the difference spectra including the protonated state of Asp396. However, we detected the prominent loss of β-sheet structure by illumination only in the presence of adenosine triphosphate (ATP). We conclude that the bound ATP stabilizes these light-induced changes in secondary structure to ensure a physiological lifetime compatible with signaling by plant cryptochrome.
In this work, we present a combination of a continuous flow reactor with in situ monitoring of the monomer conversion in a precipitation polymerization. The flow reactor is equipped with a preheating ...area for the synthesis of thermoresponsive microgels, based on
N
-isopropylacrylamide (NIPAM). The reaction progress is monitored with in situ FTIR spectroscopy. The monomer conversion at defined residence times is determined from absorbance spectra of the reaction solutions by linear combination with reference spectra of the stock solution and the purified microgel. The reconstruction of the spectra appears to be in good agreement with experimental data in the range of 1710 to 1530 cm
− 1
, in which prominent absorption bands are used as probes for the monomer and the polymer. With increasing residence time, we observed a decrease in intensity of the
ν
(C=C) vibration, originating from the monomer, while the
ν
(C=O) vibration is shifted to higher frequencies by polymerization. Differences between the determined
inline
conversion kinetics and
offline
growth kinetics, determined by photon correlation spectroscopy (PCS), are discussed in terms of diffusion and point to a crucial role of mixing in precipitation polymerizations.
Graphical Abstract
Continuous flow syntheses of acrylamide-based microgels in combination with in situ FTIR spectroscopy and offline particle characterization reveal information about the reaction progress.
Algae are photosynthetic eukaryotic (micro-)organisms, lacking roots, leaves, and other organs that are typical for land plants. They live in freshwater, marine, or terrestrial habitats. Together ...with the cyanobacteria they contribute to about half of global carbon fixation. As primary producers, they are at the basis of many food webs and they are involved in biogeochemical processes. Algae are evolutionarily distinct and are derived either by primary (e.g., green and red algae) or secondary endosymbiosis (e.g., diatoms, dinoflagellates, and brown algae). Light is a key abiotic factor needed to maintain the fitness of algae as it delivers energy for photosynthesis, regulates algal cell- and life cycles, and entrains their biological clocks. However, excess light can also be harmful, especially in the ultraviolet range. Among the variety of receptors perceiving light information, the cryptochromes originally evolved as UV-A and blue-light receptors and have been found in all studied algal genomes so far. Yet, the classification, biophysical properties, wavelength range of absorbance, and biological functions of cryptochromes are remarkably diverse among algal species, especially when compared to cryptochromes from land plants or animals.
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