Scientific publications

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.

Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

A novel approach for controlling translation initiation in mammalian cells is demonstrated based on the conditional attachment of eukaryotic translation initiation factor-binding proteins to the 3′ UTR of mRNAs via small molecule-, light-, or protein-responsive interactions. The technology overcomes limitations of previously used transcription-based switches and was shown to be functional in managing diabetes or tumor growth in preclinical animal models.

The site-specific and covalent conjugation of proteins on solid supports and in hydrogels is the basis for the synthesis of biohybrid materials offering broad applications. Current methods for conjugating proteins to desired targets are often challenging due to unspecific binding, unstable (noncovalent) coupling, or expensive and difficult-to-synthesize ligand molecules. Here, is presented PenTag, an approach for the bioorthogonal, highly specific, and covalent conjugation of a protein to its ligand for various applications in materials sciences. Penicillin-binding protein 3 (PBP3) is engineered and shows that this protein can be used for the stable and spontaneous conjugation of proteins to dyes, polymers, or solid supports. PenTag as a crosslinking tool is applied for synthesizing stimuli-responsive hydrogels or for the development of a biohybrid material system performing computational operations emulating a 4:2 encoder. Based on this broad applicability and the use of a small, cheap, and easy-to-functionalize ligand and a stable, soluble recombinant protein, is seen PenTag as a versatile approach toward biohybrid material synthesis.

Surface-grafted polymers can reduce friction between solids in liquids by compensating the normal load with osmotic pressure, but they can also contribute to friction when fluctuating polymers entangle with the sliding counter face. We have measured forces acting on a single fluctuating double-stranded DNA polymer, which is attached to the tip of an atomic force microscope and interacts intermittently with nanometer-scale methylated pores of a self-assembled polystyrene-block-poly(4-vinylpyridine) membrane. Rare binding of the polymer into the pores is followed by a stretching of the polymer between the laterally moving tip and the surface and by a force-induced detachment. We present results for the velocity dependence of detachment forces and of attachment frequency and discuss them in terms of rare excursions of the polymer beyond its equilibrium configuration.

Synthetic biology applies concepts from electrical engineering and information processing to endow cells with computational functionality. Transferring the underlying molecular components into materials and wiring them according to topologies inspired by electronic circuit boards has yielded materials systems that perform selected computational operations. However, the limited functionality of available building blocks is restricting the implementation of advanced information-processing circuits into materials. Here, a set of protease-based biohybrid modules the bioactivity of which can either be induced or inhibited is engineered. Guided by a quantitative mathematical model and following a design-build-test-learn (DBTL) cycle, the modules are wired according to circuit topologies inspired by electronic signal decoders, a fundamental motif in information processing. A 2-input/4-output binary decoder for the detection of two small molecules in a material framework that can perform regulated outputs in form of distinct protease activities is designed. The here demonstrated smart material system is strongly modular and can be used for biomolecular information processing for example in advanced biosensing or drug delivery applications.

Osteoporosis is a silent bone disease and a growing health issue. Despite recent progress in diagnosis and treatment, effective therapeutic strategies are still needed. One of the possible solutions is the implantation of engineered drug-releasing scaffolds at the disease site. To boost this approach further, we aimed to develop printable materials (the inks) for the construction of patient-specific 3D scaffolds with drug-release capability. The inks were composed of chitosan – a natural osteoinductive polysaccharide, nanohydroxyapatite – a natural bone matrix ingredient improving mechanical properties, sodium alendronate – a bioactive drug, and hydroxyethyl-cellulose – a filler improving the printability. Printed scaffolds were crosslinked with citric acid or KOH. After coating with collagen and gelatin, they demonstrated biocompatibility with the adipose-derived mesenchymal stem cells and MG-63 cell line. They also showed a sustained release of alendronate for 50 days, causing a significant reduction in the expression of Cathepsin K, an osteoclast-specific gene marker, which indicates the osteoclast-inhibiting capacity of the coated scaffolds. This work demonstrates the potential of developed printable materials to find applications as cell and drug carriers for the treatment of osteoporosis.

Probiotic bacteria, such as Lactobacilli, have been shown to elicit beneficial effects in various tissue regeneration applications. However, their formulation as living bacteria is challenging, and their therapeutic use as proliferating microorganisms is especially limited in immunocompromised patients. Here, we propose a new therapeutic avenue to circumvent these shortcomings by developing a bacteriomimetic hydrogel based on membrane vesicles (MVs) produced by Lactobacilli. We coupled MVs from Lactobacillus plantarum and Lactobacillus casei, respectively, to the surface of synthetic microparticles, and embedded those bacteriomimetics into a pharmaceutically applicable hydrogel matrix. The wound microenvironment changes during the wound healing process, including adaptions of the pH and changes of the oxygen supply. We thus performed proteomic characterization of the MVs harvested under different culture conditions and identified characteristic proteins related to the biological effect of the probiotics in every culture state. In addition, we highlight a number of unique proteins expressed and sorted into the MVs for every culture condition. Using different in vitro models, we demonstrated that increased cell migration and anti-inflammatory effects of the bacteriomimetic microparticles were dependent on the culture condition of the secreting bacteria. Finally, we demonstrated the bacteriomimetic hydrogel's ability to improve healing in an in vivo mouse full-thickness wound model. Our results create a solid basis for the future application of probiotic-derived vesicles in the treatment of inflammatory dispositions and stimulates the initiation of further preclinical trials.

The addition of water to native cellulose/1-ethyl-3-methylimidazolium acetate solutions catalyzes the formation of gels, where polymer chain–chain intermolecular associations act as cross-links. However, the relationship between water content (Wc), polymer concentration (Cp), and gel strength is still missing. This study provides the fundamentals to design water-induced gels. First, the sol–gel transition occurs exclusively in entangled solutions, while in unentangled ones, intramolecular associations hamper interchain cross-linking, preventing the gel formation. In entangled systems, the addition of water has a dual impact: at low water concentrations, the gel modulus is water-independent and controlled by entanglements. As water increases, more cross-links per chain than entanglements emerge, causing the modulus of the gel to scale as Gp ∼ Cp2Wc3.0±0.2. Immersing the solutions in water yields hydrogels with noncrystalline, aggregate-rich structures. Such water–ionic liquid exchange is examined via Raman, FTIR, and WAXS. Our findings provide avenues for designing biogels with desired rheological properties.