660 Chemische Verfahrenstechnik
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- Cell-free expression (1)
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Institute
Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of 15N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative “Stablelabel” containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in “Stablelabel” lysates. By taking advantage of ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.
Photoacids attract increasing scientific attention, as they are valuable tools to spatiotemporally control proton-release reactions and pH values of solutions. We present the first time-resolved spectroscopic study of the excited state and proton-release dynamics of prominent merocyanine representatives. Femtosecond transient absorption measurements of a pyridine merocyanine with two distinct protonation sites revealed dissimilar proton-release mechanisms: one site acts as a photoacid generator as its pKa value is modulated in the ground state after photoisomerization, while the other functions as an excited state photoacid which releases its proton within 1.1 ps. With a pKa drop of 8.7 units to −5.5 upon excitation, the latter phenolic site is regarded a super-photoacid. The 6-nitro derivative exhibits only a phenolic site with similar, yet slightly less photoacidic characteristics and both compounds transfer their proton to methanol and ethanol. In contrast, for the related 6,8-dinitro compound an intramolecular proton transfer to the ortho-nitro group is suggested that is involved in a rapid relaxation into the ground state.
Polymorphic G-quadruplex (G4) secondary DNA structures have received increasing attention in medicinal chemistry owing to their key involvement in the regulation of the maintenance of genomic stability, telomere length homeostasis and transcription of important proto-oncogenes. Different classes of G4 ligands have been developed for the potential treatment of several human diseases. Among them, the carbazole scaffold with appropriate side chain appendages has attracted much interest for designing G4 ligands. Because of its large and rigid π-conjugation system and ease of functionalization at three different positions, a variety of carbazole derivatives have been synthesized from various natural or synthetic sources for potential applications in G4-based therapeutics and biosensors. Herein, we provide an updated close-up of the literatures on carbazole-based G4 ligands with particular focus given on their detailed binding insights studied by NMR spectroscopy. The structure-activity relationships and the opportunities and challenges of their potential applications as biosensors and therapeutics are also discussed. This review will provide an overall picture of carbazole ligands with remarkable G4 topological preference, fluorescence properties and significant bioactivity; portraying carbazole as a very promising scaffold for assembling G4 ligands with a range of novel functional applications.
Phytochrome photoreceptors operate via photoisomerization of a bound bilin chromophore. Their typical architecture consists of GAF, PAS and PHY domains. Knotless phytochromes lack the PAS domain, while retaining photoconversion abilities, with some being able to photoconvert with just the GAF domain. Therefore, we investigated the ultrafast photoisomerization of the Pr state of a knotless phytochrome to reveal the effect of the PHY domain and its “tongue” region on the transduction of the light signal. We show that the PHY domain does not affect the initial conformational dynamics of the chromophore. However, it significantly accelerates the consecutively induced reorganizational dynamics of the protein, necessary for the progression of the photoisomerization. Consequently, the PHY domain keeps the bilin and its binding pocket in a more reactive conformation, which decreases the extent of protein reorganization required for the chromophore isomerization. Thereby, less energy is lost along nonproductive reaction pathways, resulting in increased efficiency.
The single crystal growth of 19 different intermetallic compounds within the LnT2X2 family (with Ln = lanthanides, T = Co, Ru, Rh, Ir, and X = Si, P) is presented, by employing a high-temperature metal-flux technique. The habitus of the obtained crystals is platelet-like with the crystallographic c direction perpendicular to the surface and with individual masses between 1 and 100 mg. The magnetic properties of these crystals are characterized by magnetization, heat-capacity, and resistivity measurements. These crystals form the materials basis for a thorough study of exciting surface properties by angle-resolved photoemission spectroscopy.