Entwicklung und Anwendung der Infrarotdifferenzspektroskopie an Blaulichtrezeptoren in Zellen

Gött-Zink L. Entwicklung und Anwendung der Infrarotdifferenzspektroskopie an Blaulichtrezeptoren in Zellen. Bielefeld: Universität Bielefeld; 2021

Plant Cryptochromes Illuminated: A Spectroscopic Perspective on the Mechanism

Gött-Zink L, Kottke T. Plant Cryptochromes Illuminated: A Spectroscopic Perspective on the Mechanism. Frontiers in Chemistry. 2021;9: 780199.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

Resolving Structural Changes of Photoreceptors in Living Escherichia coli via In-cell Infrared Difference Spectroscopy

Gött-Zink L, Klocke J, Kottke T. Resolving Structural Changes of Photoreceptors in Living Escherichia coli via In-cell Infrared Difference Spectroscopy. Bio-protocol . 2021;11(3):e3909

Time-resolved infrared difference spectroscopy in cells: Response of the basic region leucine zipper of aureochrome

Gött-Zink L, Baum E, Kottke T. Time-resolved infrared difference spectroscopy in cells: Response of the basic region leucine zipper of aureochrome. Frontiers in Physics. 2023;11: 1150671.Aureochromes are light, oxygen, voltage (LOV) proteins and central blue-light receptors in algae acting as light-gated transcription factors. The C-terminal LOV domain mediates blue-light recognition and the basic region leucine zipper (bZIP) domain binds a specific DNA motif as effector. LOV domains from aureochromes have been successfully applied in optogenetic tools. The light-induced response of aureochromes has been studied by a variety of biophysical techniques, but the mechanism of signal progression from LOV to bZIP remains unclear. We studied the bZIP-LOV module of aureochrome1a from the diatomPhaeodactylum tricornutumusing time-resolved rapid-scan FTIR difference spectroscopy. Time-resolved difference spectra of bZIP-LOVin vitrorevealed a time constant of 5 s for the formation of a light state dimer of the LOV domains and the concomitant loss of α-helical elements in the bZIP domain. To verify these observations in a near-native environment, in-cell infrared difference spectroscopy (ICIRD) was extended from a steady state to a time-resolved technique using LOV domains in bacterial cells. We established a time-resolved in-cell method with a resolution of 7.6 ms after the laser pulse. Using this technique, the response of bZIP-LOV was followed in living bacterial cells and the light-induced partial unfolding of bZIP was confirmed to take place in cells in a similar time range asin vitro. These results provide structural and kinetic insights into the signaling mechanism of aureochromes. The slow response points to an association of LOV to bZIP in the dark state prior to activation

In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells

Gött-Zink L, Klocke J, Bögeholz LAK, Kottke T. In-cell infrared difference spectroscopy of LOV photoreceptors reveals structural responses to light altered in living cells. Journal of Biological Chemistry. 2020;295(33):11729-11741.Proteins are usually studied in well-defined buffer conditions, which differ substantially from those within a host cell. In some cases, the intracellular environment has an impact on the mechanism, which might be missed by in vitro experiments. Infrared difference spectroscopy previously has been applied to study the light-induced response of photoreceptors and photoenzymes in vitro Here, we established the in-cell infrared difference (ICIRD) spectroscopy in the transmission and attenuated total reflection (ATR) configuration to investigate the light-induced response of soluble proteins in living bacterial cells. ICIRD spectroscopy on the light, oxygen, or voltage (LOV) domains of the blue light receptors aureochrome and phototropin revealed a suppression of the response of specific secondary structure elements, indicating that the intracellular environment affects LOV photoreceptor mechanisms in general. Moreover, in-cell fluorescence spectroscopy disclosed that the intracellular environment slows down the recovery of the light-induced flavin adduct. Segment-resolved ICIRD spectroscopy on basic-region leucine zipper (bZIP)-LOV of aureochrome 1a from the diatom Phaeodactylum tricornutum indicated a signal progression from the LOV sensor to the bZIP effector independent of unfolding of the connecting A'alpha-helix, an observation that stood in contrast to in vitro results. This deviation was recapitulated in vitro by emulating the intracellular environment through addition of the crowding agent bovine serum albumin, but not by sucrose polymers. We conclude that ICIRD spectroscopy is a non-invasive, label-free approach for assessing conformational changes in receptors in living cells at ambient conditions. As demonstrated, these near-native responses may deviate from the mechanisms established under in vitro conditions

C-Terminal Extension of a Plant Cryptochrome Dissociates from the β-Sheet of the Flavin-Binding Domain

Gött-Zink L, Toschke AL, Petersen J, Mittag M, Kottke T. C-Terminal Extension of a Plant Cryptochrome Dissociates from the β-Sheet of the Flavin-Binding Domain. The Journal of Physical Chemistry Letters. 2021;12:5558-5563

Thermoresponsive Microgel-Based Free-Standing Membranes: Influence of Different Microgel Cross-Linkers on Membrane Function

Dirksen M, Fandrich P, Gött-Zink L, Cremer J, Anselmetti D, Hellweg T. Thermoresponsive Microgel-Based Free-Standing Membranes: Influence of Different Microgel Cross-Linkers on Membrane Function. Langmuir. 2022: acs.langmuir.1c02195

Tongue Refolding in the Knotless Cyanobacterial Phytochrome All2699

Xu Q-Z, Gött-Zink L, Gärtner W, Zhao K-H, Kottke T. Tongue Refolding in the Knotless Cyanobacterial Phytochrome All2699. Biochemistry. 2020;59(22):2047-2054

Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates

Karsten L, Gött-Zink L, Schmitz J, et al. Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates. Biosensors. 2022;12(5): 271.pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK′ = 6.6) and EGF-CoGFP-mCRISPRed (pK′ = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein–drug conjugates in cancer therapy

Biphasic Formation of 2D Nanomembranes by Photopolymerization of Diacetylene Lipids as Revealed by Infrared Difference Spectroscopy

Gilzer D, Hillmann R, Gött-Zink L, et al. Biphasic Formation of 2D Nanomembranes by Photopolymerization of Diacetylene Lipids as Revealed by Infrared Difference Spectroscopy. Langmuir. 2019;35(29):9343-9351