Franz-Josef Schmitt

In this study, melanoidins formed from fructosylglycine and heated mixtures of glycine and glucose were analyzed and compared using spectroscopic techniques including UV/Vis, FTIR, EPR, NMR, as well as elemental analysis (EA). EA revealed that melanoidin formed from fructosylglycine incorporates a higher proportion of glycine compared to melanoidin produced through the direct reaction of glycine and glucose upon heating. FTIR spectra identified carbonyl or carboxyl groups with distinct bands at ~ 1749–1759 cm⁻¹, contributing to the extended π-electron system observed at 170–200 ppm in NMR spectra. EPR measurements demonstrated a higher abundance of unpaired electrons in fructosylglycine-derived melanoidin. The UV/Vis, FTIR, and NMR data indicated that the backbones of fructosylglycine-derived melanoidins contain a greater number of conjugated π bonds. Therefore, we conclude that the melanoidin skeleton synthesized from fructosylglycine includes more amino acid residues, owing to enhanced activation of nitrogen in the secondary amine of fructosylglycine compared to the primary amine of glycine. EPR results further reveal a positive correlation between melanoidin absorbance spectra, the size of their π-electron system, and antioxidant activity. These findings suggest that the nucleophilic attack of glycine’s amino group on glucose’s carbonyl group is facilitated in secondary amines, indicating a potential pathway to enhance melanoidin formation by incorporating secondary amines during food processing.

Fluorescing proteins emitting in the near-infrared region are of high importance in various fields of biomedicine and applied life sciences. Promising candidates are phytochromes that can be engineered to a small size and genetically attached to a target system for in vivo monitoring. Here, we have investigated two of these minimal single-domain phytochromes, miRFP670nano3 and miRFP718nano, aiming at a better understanding of the structural parameters that control the fluorescence properties of the covalently bound biliverdin (BV) chromophore. On the basis of resonance Raman and time-resolved fluorescence spectroscopy, it is shown that in both proteins, BV is deprotonated at one of the inner pyrrole rings (B or C). This protonation pattern, which is unusual for tetrapyrroles in proteins, implies an equilibrium between a B- and C-protonated tautomer. The dynamics of the equilibrium are slow compared to the fluorescence lifetime in miRFP670nano3 but much faster in miRFP718nano, both in the ground and excited states. The different rates of proton exchange are most likely due to the different structural dynamics of the more rigid and more flexible chromophore in miRFP670nano3 and miRFP718nano, respectively. We suggest that these structural properties account for the quite different fluorescent quantum yields of both proteins.

Oxygenic photosynthesis in Halomicronema hongdechloris, one of a series of cyanobacteria producing red-shifted Chl f, is adapted to varying light conditions by a range of diverse processes acting over largely different time scales. Acclimation to far-red light (FRL) above 700 nm over several days is mirrored by reversible changes in the Chl f content. In several cyanobacteria that undergo FRL photoacclimation, Chl d and Chl f are directly involved in excitation energy transfer in the antenna system, form the primary donor in photosystem I (PSI), and are also involved in electron transfer within photosystem II (PSII), most probably at the Chl_D1 position, with efficient charge transfer happening with comparable kinetics to reaction centers containing Chl a. In H. hongdechloris, the formation of Chl f under FRL comes along with slow adaptive proteomic shifts like the rebuilding of the D1 complex on the time scale of days. On shorter time scales, much faster adaptation mechanisms exist involving the phycobilisomes (PBSs), which mainly contain allophycocyanin upon adaptation to FRL. Short illumination with white, blue, or red light leads to reactive oxygen species-driven mobilization of the PBSs on the time scale of seconds, in effect recoupling the PBSs with Chl f-containing PSII to re-establish efficient excitation energy transfer within minutes. In summary, H. hongdechloris reorganizes PSII to act as a molecular heat pump lifting excited states from Chl f to Chl a on the picosecond time scale in combination with a light-driven PBS reorganization acting on the time scale of seconds to minutes depending on the actual light conditions. Thus, structure–function relationships in photosynthetic energy and electron transport in H. hongdechloris including long-term adaptation processes cover 10⁻¹² to 10⁶ seconds, i.e., 18 orders of magnitude in time.

Synthetic biology, an interdisciplinary field merging biology, engineering, and computer science, holds significant promise but also gives rise to ethical and safety issues and concerns. Effective communication of scientific concepts is essential to bridge the gap between the scientific community and the general public. Here we present four communication strategies from our own experience that could help address this gap: (i) the teaching module “iGEM-Synthetic Biology”: Students at Technische Universität Berlin (TU Berlin) engage in synthetic biology projects, showcasing their work in a competition format that includes the creation of videos and websites. (ii) Long Night of the Sciences: Students and supervisors from the international Genetically Engineered Machine (iGEM) module at TU Berlin share their results with presentations, experiments, and quiz games during this public event. (iii) Theatre play: Festival für Freunde e.V. has developed a play titled “Life from the Toolbox” to explain genetic modification and synthetic biology for the audience. The play incorporates readings and educational videos. (iv) Heinz-Bethge-Foundation electron microscopy school lab: This laboratory utilizes hands-on experiments with microscopes, including electron microscopes, to visually explain intricate scientific concepts in physics, biology, and synthetic biology. It encourages high school students to delve deeper into the realm of science. These four initiatives represent a communication strategy that resonates with diverse audiences and is suitable to cover the public as a target group independent from their prior knowledge of the scientific background.

Halomicronema hongdechloris, the first cyanobacterium reported to produce the red-shifted chlorophyll f (Chl f) upon acclimation to far-red light, demonstrates remarkable adaptability to diverse light conditions. The photosystem II (PS II) of this organism undergoes reversible changes in its Chl f content, ranging from practically zero under white-light culture conditions to a Chl f: Chl a ratio of up to 1:8 when exposed to far-red light (FRL) of 720–730 nm for several days. Our ps time- and wavelength-resolved fluorescence data obtained after excitation of living H. hongdechloris cells indicate that the Soret band of a far-red (FR) chlorophyll involved in charge separation absorbs around 470 nm. At 10 K, the fluorescence decay at 715–720 nm is still fast with a time constant of 165 ps indicating an efficient electron tunneling process. There is efficient excitation energy transfer (EET) from 715–720 nm to 745 nm with the latter resulting from FR Chl f, which mainly functions as light-harvesting pigment upon adaptation to FRL. From there, excitation energy reaches the primary donor in the reaction center of PS II with an energetic uphill EET mechanism inducing charge transfer. The fluorescence data are well explained with a secondary donor P_D1 represented by a red-shifted Chl a molecule with characteristic fluorescence around 715 nm and a more red-shifted FR Chl f with fluorescence around 725 nm as primary donor at the Chl_D1 or P_D2 position.

The pH dependence of the absorption and (time-resolved) fluorescence of two red-shifted fluorescent proteins, mCardinal and mNeptune, was investigated. Decay-associated spectra were measured following fluorescence excitation at 470 nm in PBS buffer with a pH that ranged from 5.5 to 8.0. The fluorescence of both proteins shows two different decay components. mCardinal exhibits an increase in the long-lived fluorescence component with acidification from 1.34 ns at pH 8.0 to 1.62 ns at pH 5.5. An additional fast decay component with 0.64 ns at pH 8.0 up to 1.1 ns at pH 5.5 was found to be blue-shifted compared to the long-lived component. The fluorescence lifetime of mNeptune is insensitive to pH. DAS of mCardinal were simulated assuming a coupled two-level system to describe the ¹S state of the chromophore within two different conformations of the protein. MD simulations were conducted to correlate the experimentally observed pH-induced change in the lifetime in mCardinal with its molecular properties. While the chromophores of both protein variants are stabilized by the same number of hydrogen bonds, it was found that the chromophore in mCardinal exhibits more water contacts compared to mNeptune. In mCardinal, interaction between the chromophore and Glu-145 is reduced as compared to mNeptune, but interaction with Thr-147 which is Ser-147 in mNeptune is stronger in mCardinal. Therefore, the dynamics of the excited-state proton transfer (ESPT) might be different in mCardinal and mNeptune. The pH dependency of ESPT is suggested as a key mechanism for pH sensitivity.

Heat-triggered enhancement of contrast in pump–probe photoacoustic measurements is reported. Changes in polarity lead to considerable effects in photophysical properties of fluorescently labelled single-chain nanoparticles (SCNPs).

Halomicronema hongdechloris, the first cyanobacterium reported to produce the red-shifted chlorophyll f (Chl f) upon acclimation to far-red light, demonstrates remarkable adaptability to diverse light conditions. The photosystem II (PS II) of this organism undergoes reversible changes in its Chl f content, with levels ranging from practically zero under white-light culture conditions to a Chl f :Chl a ratio of up to 1:8 when exposed to far-red light (FRL) in the 720–730 nm range for several days. Our ps time- and wavelength-resolved fluorescence data obtained after excitation of living H. hongdechloris cells indicate that the Soret band of a far-red (FR) chlorophyll involved in charge separation absorbs at 470 nm. At 10 K, the fluorescence decay at 715–720 nm is still fast with a time constant of 165 ps indicating an efficient electron tunneling process. However, additionally, there is efficient excitation energy transfer (EET) from 715–720 nm to 745 nm with the latter resulting from FR Chl f, which mainly functions as light-harvesting pigment upon adaptation to FRL. From there, excitation energy is efficiently transferred towards the primary donor in the reaction center of PS II with an energetic uphill EET mechanism inducing charge transfer. The fluorescence data is well explained with a secondary donor P_D1 represented by a red-shifted Chl a molecule with characteristic fluorescence around 715 nm and a more red-shifted FR Chl f with fluorescence around 725 nm as primary donor at the Chl_D1 or P_D2 position.

Controlling the internal structures of single‐chain nanoparticles (SCNPs) is an important factor for their targeted chemical design and synthesis, especially in view of nanosized compartments presenting different local environments as a main feature to control functionality. We here design SCNPs bearing near‐infrared fluorescent dyes embedded in hydrophobic compartments for use as contrast agents in pump–probe photoacoustic (PA) imaging, displaying improved properties by the location of the dye in the hydrophobic particle core. Compartment formation is controlled via single‐chain collapse and subsequent crosslinking of an amphiphilic polymer using external crosslinkers in reaction media of adjustable polarity. Different SCNPs with hydrodynamic diameters of 6‐12 nm bearing adjustable label densities are synthesized. It is found that the specific conditions for single‐chain collapse have a major impact on the formation of the desired core–shell structure, in turn adjusting the internal nanocompartments together with the formation of excitonic dye couples, which in turn increase their fluorescence lifetime and PA signal generation. SCNPs with the dye molecules accumulate at the core also show a nonlinear PA response as a function of pulse energy—a property that can be exploited as a contrast mechanism in molecular PA tomography.