TY - GEN A1 - Fichtner, Franziska A1 - Olas, Justyna Jadwiga A1 - Feil, Regina A1 - Watanabe, Mutsumi A1 - Krause, Ursula A1 - Hoefgen, Rainer A1 - Stitt, Mark A1 - Lunn, John Edward T1 - Functional features of Trehalose-6-Phosphate Synthase 1 BT - an essential enzyme in Arabidopsis T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Tre6P synthesis by TPS1 is essential for embryogenesis and postembryonic growth in Arabidopsis, and appropriate Suc signaling by Tre6P is dependent on the noncatalytic domains of TPS1. In Arabidopsis (Arabidopsis thaliana), TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) catalyzes the synthesis of the sucrose-signaling metabolite trehalose 6-phosphate (Tre6P) and is essential for embryogenesis and normal postembryonic growth and development. To understand its molecular functions, we transformed the embryo-lethal tps1-1 null mutant with various forms of TPS1 and with a heterologous TPS (OtsA) from Escherichia coli, under the control of the TPS1 promoter, and tested for complementation. TPS1 protein localized predominantly in the phloem-loading zone and guard cells in leaves, root vasculature, and shoot apical meristem, implicating it in both local and systemic signaling of Suc status. The protein is targeted mainly to the nucleus. Restoring Tre6P synthesis was both necessary and sufficient to rescue the tps1-1 mutant through embryogenesis. However, postembryonic growth and the sucrose-Tre6P relationship were disrupted in some complementation lines. A point mutation (A119W) in the catalytic domain or truncating the C-terminal domain of TPS1 severely compromised growth. Despite having high Tre6P levels, these plants never flowered, possibly because Tre6P signaling was disrupted by two unidentified disaccharide-monophosphates that appeared in these plants. The noncatalytic domains of TPS1 ensure its targeting to the correct subcellular compartment and its catalytic fidelity and are required for appropriate signaling of Suc status by Tre6P. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1432 KW - cyanobacterial sucrose-phosphatase KW - trehalose 6-phosphate KW - vegetative growth KW - crystal-structure KW - gene-expression KW - thaliana KW - metabolism KW - phosphorylation KW - reveals KW - proteins Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-516532 SN - 1866-8372 IS - 6 ER - TY - JOUR A1 - Fichtner, Franziska A1 - Olas, Justyna Jadwiga A1 - Feil, Regina A1 - Watanabe, Mutsumi A1 - Krause, Ursula A1 - Hoefgen, Rainer A1 - Stitt, Mark A1 - Lunn, John Edward T1 - Functional features of Trehalose-6-Phosphate Synthase 1 BT - an essential enzyme in Arabidopsis JF - The Plant Cell N2 - Tre6P synthesis by TPS1 is essential for embryogenesis and postembryonic growth in Arabidopsis, and appropriate Suc signaling by Tre6P is dependent on the noncatalytic domains of TPS1. In Arabidopsis (Arabidopsis thaliana), TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) catalyzes the synthesis of the sucrose-signaling metabolite trehalose 6-phosphate (Tre6P) and is essential for embryogenesis and normal postembryonic growth and development. To understand its molecular functions, we transformed the embryo-lethal tps1-1 null mutant with various forms of TPS1 and with a heterologous TPS (OtsA) from Escherichia coli, under the control of the TPS1 promoter, and tested for complementation. TPS1 protein localized predominantly in the phloem-loading zone and guard cells in leaves, root vasculature, and shoot apical meristem, implicating it in both local and systemic signaling of Suc status. The protein is targeted mainly to the nucleus. Restoring Tre6P synthesis was both necessary and sufficient to rescue the tps1-1 mutant through embryogenesis. However, postembryonic growth and the sucrose-Tre6P relationship were disrupted in some complementation lines. A point mutation (A119W) in the catalytic domain or truncating the C-terminal domain of TPS1 severely compromised growth. Despite having high Tre6P levels, these plants never flowered, possibly because Tre6P signaling was disrupted by two unidentified disaccharide-monophosphates that appeared in these plants. The noncatalytic domains of TPS1 ensure its targeting to the correct subcellular compartment and its catalytic fidelity and are required for appropriate signaling of Suc status by Tre6P. KW - cyanobacterial sucrose-phosphatase KW - trehalose 6-phosphate KW - vegetative growth KW - crystal-structure KW - gene-expression KW - thaliana KW - metabolism KW - phosphorylation KW - reveals KW - proteins Y1 - 2020 U6 - https://doi.org/10.1105/tpc.19.00837 SN - 0032-0781 SN - 1471-9053 VL - 32 IS - 6 SP - 1949 EP - 1972 PB - Oxford University Press CY - Oxford ER - TY - JOUR A1 - Deutschmann, Claudia A1 - Roggenbuck, Dirk A1 - Schierack, Peter A1 - Rödiger, Stefan T1 - Autoantibody testing by enzyme-linked immunosorbent assay-a case in which the solid phase decides on success and failure JF - Heliyon N2 - Background: The enzyme-linked immunosorbent assay (ELISA) is an indispensable tool for clinical diagnostics to identify or differentiate diseases such as autoimmune illnesses, but also to monitor their progression or control the efficacy of drugs. One use case of ELISA is to differentiate between different states (e.g. healthy vs. diseased). Another goal is to quantitatively assess the biomarker in question, like autoantibodies. Thus, the ELISA technology is used for the discovery and verification of new autoantibodies, too. Of key interest, however, is the development of immunoassays for the sensitive and specific detection of such biomarkers at early disease stages. Therefore, users have to deal with many parameters, such as buffer systems or antigen-autoantibody interactions, to successfully establish an ELISA. Often, fine-tuning like testing of several blocking substances is performed to yield high signal-to-noise ratios.
Methods: We developed an ELISA to detect IgA and IgG autoantibodies against chitinase-3-like protein 1 (CHI3L1), a newly identified autoantigen in inflammatory bowel disease (IBD), in the serum of control and disease groups (n = 23, respectively). Microwell plates with different surface modifications (PolySorp and MaxiSorp coating) were tested to detect reproducibility problems.
Results: We found a significant impact of the surface properties of the microwell plates. IgA antibody reactivity was significantly lower, since it was in the range of background noise, when measured on MaxiSorp coated plates (p < 0.0001). The IgG antibody reactivity did not differ on the diverse plates, but the plate surface had a significant influence on the test result (p = 0.0005).
Conclusion: With this report, we want to draw readers' attention to the properties of solid phases and their effects on the detection of autoantibodies by ELISA. We want to sensitize the reader to the fact that the choice of the wrong plate can lead to a false negative test result, which in turn has serious consequences for the discovery of autoantibodies. KW - biochemistry KW - coatings KW - surface chemistry KW - immunology KW - proteins KW - laboratory medicine KW - clinical research KW - enzyme-linked immunosorbent KW - assay KW - biomarker discovery KW - reproducibility KW - solid-phase KW - autoantibody Y1 - 2020 U6 - https://doi.org/10.1016/j.heliyon.2020.e03270 SN - 2405-8440 VL - 6 IS - 1 PB - Elsevier CY - London [u.a.] ER - TY - THES A1 - Förste, Stefanie T1 - Assemblierung von Proteinkomplexen in vitro und in vivo T1 - Assembly of protein complexes in vitro and in vivo N2 - Proteine sind an praktisch allen Prozessen in lebenden Zellen maßgeblich beteiligt. Auch in der Biotechnologie werden Proteine in vielfältiger Weise eingesetzt. Ein Protein besteht aus einer Kette von Aminosäuren. Häufig lagern sich mehrere dieser Ketten zu größeren Strukturen und Funktionseinheiten, sogenannten Proteinkomplexen, zusammen. Kürzlich wurde gezeigt, dass eine Proteinkomplexbildung bereits während der Biosynthese der Proteine (co-translational) stattfinden kann und nicht stets erst danach (post-translational) erfolgt. Da Fehlassemblierungen von Proteinen zu Funktionsverlusten und adversen Effekten führen, ist eine präzise und verlässliche Proteinkomplexbildung sowohl für zelluläre Prozesse als auch für biotechnologische Anwendungen essenziell. Mit experimentellen Methoden lassen sich zwar u.a. die Stöchiometrie und die Struktur von Proteinkomplexen bestimmen, jedoch bisher nicht die Dynamik der Komplexbildung auf unterschiedlichen Zeitskalen. Daher sind grundlegende Mechanismen der Proteinkomplexbildung noch nicht vollständig verstanden. Die hier vorgestellte, auf experimentellen Erkenntnissen aufbauende, computergestützte Modellierung der Proteinkomplexbildung erlaubt eine umfassende Analyse des Einflusses physikalisch-chemischer Parameter auf den Assemblierungsprozess. Die Modelle bilden möglichst realistisch die experimentellen Systeme der Kooperationspartner (Bar-Ziv, Weizmann-Institut, Israel; Bukau und Kramer, Universität Heidelberg) ab, um damit die Assemblierung von Proteinkomplexen einerseits in einem quasi-zweidimensionalen synthetischen Expressionssystem (in vitro) und andererseits im Bakterium Escherichia coli (in vivo) untersuchen zu können. Mit Hilfe eines vereinfachten Expressionssystems, in dem die Proteine nur an die Chip-Oberfläche, aber nicht aneinander binden können, wird das theoretische Modell parametrisiert. In diesem vereinfachten in-vitro-System durchläuft die Effizienz der Komplexbildung drei Regime – ein bindedominiertes Regime, ein Mischregime und ein produktionsdominiertes Regime. Ihr Maximum erreicht die Effizienz dabei kurz nach dem Übergang vom bindedominierten ins Mischregime und fällt anschließend monoton ab. Sowohl im nicht-vereinfachten in-vitro- als auch im in-vivo-System koexistieren je zwei konkurrierende Assemblierungspfade: Im in-vitro-System erfolgt die Komplexbildung entweder spontan in wässriger Lösung (Lösungsassemblierung) oder aber in einer definierten Schrittfolge an der Chip-Oberfläche (Oberflächenassemblierung); Im in-vivo-System konkurrieren hingegen die co- und die post-translationale Komplexbildung. Es zeigt sich, dass die Dominanz der Assemblierungspfade im in-vitro-System zeitabhängig ist und u.a. durch die Limitierung und Stärke der Bindestellen auf der Chip-Oberfläche beeinflusst werden kann. Im in-vivo-System hat der räumliche Abstand zwischen den Syntheseorten der beiden Proteinkomponenten nur dann einen Einfluss auf die Komplexbildung, wenn die Untereinheiten schnell degradieren. In diesem Fall dominiert die co-translationale Assemblierung auch auf kurzen Zeitskalen deutlich, wohingegen es bei stabilen Untereinheiten zu einem Wechsel von der Dominanz der post- hin zu einer geringen Dominanz der co-translationalen Assemblierung kommt. Mit den in-silico-Modellen lässt sich neben der Dynamik u.a. auch die Lokalisierung der Komplexbildung und -bindung darstellen, was einen Vergleich der theoretischen Vorhersagen mit experimentellen Daten und somit eine Validierung der Modelle ermöglicht. Der hier präsentierte in-silico Ansatz ergänzt die experimentellen Methoden, und erlaubt so, deren Ergebnisse zu interpretieren und neue Erkenntnisse davon abzuleiten. KW - Assemblierung KW - Proteine KW - Multiproteinkomplexbildung KW - co-translationale Assemblierung KW - post-translationale Assemblierung KW - Lösung KW - Oberfläche KW - Lösungsassemblierung KW - Oberflächenassemblierung KW - co-translational KW - post-translational KW - assembly KW - proteins KW - multi protein complex formation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-550742 ER - TY - GEN A1 - Walkowiak, Jacek A1 - Lu, Yan A1 - Gradzielski, Michael A1 - Zauscher, Stefan A1 - Ballauff, Matthias T1 - Thermodynamic analysis of the uptake of a protein in a spherical polyelectrolyte brush T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - A thermodynamic study of the adsorption of Human Serum Albumin (HSA) onto spherical polyelectrolyte brushes (SPBs) by isothermal titration calorimetry (ITC) is presented. The SPBs are composed of a solid polystyrene core bearing long chains of poly(acrylic acid). ITC measurements done at different temperatures and ionic strengths lead to a full set of thermodynamicbinding constants together with the enthalpies and entropies of binding. The adsorption of HSA onto SPBs is described with a two-step model. The free energy of binding Delta Gb depends only weakly on temperature because of a marked compensation of enthalpy by entropy. Studies of the adsorbed HSA by Fourier transform infrared spectroscopy (FT-IR) demonstrate no significant disturbance in the secondary structure of the protein. The quantitative analysis demonstrates that counterion release is the major driving force for adsorption in a process where proteins become multivalent counterions of the polyelectrolyte chains upon adsorption. A comparison with the analysis of other sets of data related to the binding of HSA to polyelectrolytes demonstrates that the cancellation of enthalpy and entropy is a general phenomenon that always accompanies the binding of proteins to polyelectrolytes dominated by counterion release. KW - ITC KW - spherical polyelectrolyte brushes KW - enthalpy-entropy compensation (EEC) KW - proteins KW - thermodynamics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-517307 SN - 1866-8372 IS - 1 ER - TY - JOUR A1 - Walkowiak, Jacek A1 - Lu, Yan A1 - Gradzielski, Michael A1 - Zauscher, Stefan A1 - Ballauff, Matthias T1 - Thermodynamic analysis of the uptake of a protein in a spherical polyelectrolyte brush JF - Macromolecular rapid communications N2 - A thermodynamic study of the adsorption of Human Serum Albumin (HSA) onto spherical polyelectrolyte brushes (SPBs) by isothermal titration calorimetry (ITC) is presented. The SPBs are composed of a solid polystyrene core bearing long chains of poly(acrylic acid). ITC measurements done at different temperatures and ionic strengths lead to a full set of thermodynamicbinding constants together with the enthalpies and entropies of binding. The adsorption of HSA onto SPBs is described with a two-step model. The free energy of binding Delta Gb depends only weakly on temperature because of a marked compensation of enthalpy by entropy. Studies of the adsorbed HSA by Fourier transform infrared spectroscopy (FT-IR) demonstrate no significant disturbance in the secondary structure of the protein. The quantitative analysis demonstrates that counterion release is the major driving force for adsorption in a process where proteins become multivalent counterions of the polyelectrolyte chains upon adsorption. A comparison with the analysis of other sets of data related to the binding of HSA to polyelectrolytes demonstrates that the cancellation of enthalpy and entropy is a general phenomenon that always accompanies the binding of proteins to polyelectrolytes dominated by counterion release. KW - Spherical polyelectrolyte brushes KW - proteins KW - ITC KW - thermodynamics KW - enthalpy-entropy compensation (EEC) Y1 - 2019 U6 - https://doi.org/10.1002/marc.201900421 SN - 1022-1336 SN - 1521-3927 VL - 41 IS - 1 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Semenyshyn, Rostyslav A1 - Hentschel, Mario A1 - Stanglmair, Christoph A1 - Teutsch, Tanja A1 - Tarin, Cristina A1 - Pacholski, Claudia A1 - Giessen, Harald A1 - Neubrech, Frank T1 - In vitro monitoring conformational changes of polypeptide monolayers using infrared plasmonic nanoantennas JF - Nano letters : a journal dedicated to nanoscience and nanotechnology N2 - Proteins and peptides play a predominant role in biochemical reactions of living cells. In these complex environments, not only the constitution of the molecules but also their three-dimensional configuration defines their functionality. This so-called secondary structure of proteins is crucial for understanding their function in living matter. Misfolding, for example, is suspected as the cause of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Ultimately, it is necessary to study a single protein and its folding dynamics. Here, we report a first step in this direction, namely ultrasensitive detection and discrimination of in vitro polypeptide folding and unfolding processes using resonant plasmonic nanoantennas for surface-enhanced vibrational spectroscopy. We utilize poly-l-lysine as a model system which has been functionalized on the gold surface. By in vitro infrared spectroscopy of a single molecular monolayer at the amide I vibrations we directly monitor the reversible conformational changes between α-helix and β-sheet states induced by controlled external chemical stimuli. Our scheme in combination with advanced positioning of the peptides and proteins and more brilliant light sources is highly promising for ultrasensitive in vitro studies down to the single protein level. KW - Plasmonics KW - surface-enhanced infrared absorption spectroscopy KW - proteins KW - conformational changes KW - biosensing Y1 - 2019 U6 - https://doi.org/10.1021/acs.nanolett.8b02372 SN - 1530-6984 SN - 1530-6992 VL - 19 IS - 1 SP - 1 EP - 7 PB - American Chemical Society CY - Washington ER - TY - GEN A1 - Nakamura, Moritaka A1 - Claes, Andrea R. A1 - Grebe, Tobias A1 - Hermkes, Rebecca A1 - Viotti, Corrado A1 - Ikeda, Yoshihisa A1 - Grebe, Markus T1 - Auxin and ROP GTPase signaling of polar nuclear migration in root epidermal hair cells T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Polar nuclear migration is crucial during the development of diverse eukaryotes. In plants, root hair growth requires polar nuclear migration into the outgrowing hair. However, knowledge about the dynamics and the regulatory mechanisms underlying nuclear movements in root epidermal cells remains limited. Here, we show that both auxin and Rho-of-Plant (ROP) signaling modulate polar nuclear position at the inner epidermal plasma membrane domain oriented to the cortical cells during cell elongation as well as subsequent polar nuclear movement to the outer domain into the emerging hair bulge in Arabidopsis (Arabidopsis thaliana). Auxin signaling via the nuclear AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and INDOLE ACETIC ACID7 pathway ensures correct nuclear placement toward the inner membrane domain. Moreover, precise inner nuclear placement relies on SPIKE1 Rho-GEF, SUPERCENTIPEDE1 Rho-GDI, and ACTIN7 (ACT7) function and to a lesser extent on VTI11 vacuolar SNARE activity. Strikingly, the directionality and/or velocity of outer polar nuclear migration into the hair outgrowth along actin strands also are ACT7 dependent, auxin sensitive, and regulated by ROP signaling. Thus, our findings provide a founding framework revealing auxin and ROP signaling of inner polar nuclear position with some contribution by vacuolar morphology and of actin-dependent outer polar nuclear migration in root epidermal hair cells. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 992 KW - Arabidopsis-thaliana KW - planar polarity KW - tip growth KW - morphogenesis KW - gene KW - proteins KW - dynamics KW - transformation KW - activation KW - initiation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-441278 SN - 1866-8372 IS - 992 SP - 378 EP - 391 ER - TY - JOUR A1 - Tebaldi, Marli Luiza A1 - Charan, Himanshu A1 - Mavliutova, Liliia A1 - Böker, Alexander A1 - Glebe, Ulrich T1 - Dual-Stimuli Sensitive Hybrid Materials: Ferritin-PDMAEMA by Grafting-From Polymerization JF - Macromolecular chemistry and physics N2 - The combination of stimuli-responsive polymers and proteins that can transport drugs is a promising approach for drug delivery. The formation of ferritin-poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) conjugates by atom-transfer radical polymerization from the protein macroinitiator is described. PDMAEMA is a dual-stimuli-responsive polymer and the thermo- and pH-responsive properties of the resulting conjugates are studied in detail with dynamic light scattering (DLS). Additionally, it is demonstrated that the lower critical solution temperature (LCST) of the protein-polymer conjugates can be further adjusted by the ionic strength of the solution. The conjugates are also characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry, and NMR spectroscopy. The obtained MALDI-ToF mass spectra are exceptional for protein-polymer conjugates and have not been so often reported. KW - grafting-from KW - MALDI-ToF MS KW - polymerization KW - proteins KW - responsivity Y1 - 2017 U6 - https://doi.org/10.1002/macp.201600529 SN - 1022-1352 SN - 1521-3935 VL - 218 PB - Wiley-VCH CY - Weinheim ER - TY - GEN A1 - Wessig, Pablo A1 - Hille, Carsten A1 - Kumke, Michael Uwe A1 - Meiling, Till Thomas A1 - Behrends, Nicole A1 - Eisold, Ursula T1 - Two-photon FRET pairs based on coumarin and DBD dyes N2 - The synthesis and photophysical properties of two new FRET pairs based on coumarin as a donor and DBD dye as an acceptor are described. The introduction of a bromo atom dramatically increases the two-photon excitation (2PE) cross section providing a 2PE-FRET system, which is also suitable for 2PE-FLIM. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 318 KW - resonance energy-tansfer KW - conformational-changes KW - microscopy KW - proteins KW - acid Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-394445 SP - 33510 EP - 33513 ER -