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 - 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 - 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 - 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 - THES A1 - Laux, Eva-Maria T1 - Electric field-assisted immobilization and alignment of biomolecules T1 - Immobilisierung und Ausrichtung von Biomolekülen mit elektrischen Wechselfeldern N2 - In this dissertation, an electric field-assisted method was developed and applied to achieve immobilization and alignment of biomolecules on metal electrodes in a simple one-step experiment. Neither modifications of the biomolecule nor of the electrodes were needed. The two major electrokinetic effects that lead to molecule motion in the chosen electrode configurations used were identified as dielectrophoresis and AC electroosmotic flow. To minimize AC electroosmotic flow, a new 3D electrode configuration was designed. Thus, the influence of experimental parameters on the dielectrophoretic force and the associated molecule movement could be studied. Permanent immobilization of proteins was examined and quantified absolutely using an atomic force microscope. By measuring the volumes of the immobilized protein deposits, a maximal number of proteins contained therein was calculated. This was possible since the proteins adhered to the tungsten electrodes even after switching off the electric field. The permanent immobilization of functional proteins on surfaces or electrodes is one crucial prerequisite for the fabrication of biosensors. Furthermore, the biofunctionality of the proteins must be retained after immobilization. Due to the chemical or physical modifications on the proteins caused by immobilization, their biofunctionality is sometimes hampered. The activity of dielectrophoretically immobilized proteins, however, was proven here for an enzyme for the first time. The enzyme horseradish peroxidase was used exemplarily, and its activity was demonstrated with the oxidation of dihydrorhodamine 123, a non-fluorescent precursor of the fluorescence dye rhodamine 123. Molecular alignment and immobilization - reversible and permanent - was achieved under the influence of inhomogeneous AC electric fields. For orientational investigations, a fluorescence microscope setup, a reliable experimental procedure and an evaluation protocol were developed and validated using self-made control samples of aligned acridine orange molecules in a liquid crystal. Lambda-DNA strands were stretched and aligned temporarily between adjacent interdigitated electrodes, and the orientation of PicoGreen molecules, which intercalate into the DNA strands, was determined. Similarly, the aligned immobilization of enhanced Green Fluorescent Protein was demonstrated exploiting the protein's fluorescence and structural properties. For this protein, the angle of the chromophore with respect to the protein's geometrical axis was determined in good agreement with X-ray crystallographic data. Permanent immobilization with simultaneous alignment of the proteins was achieved along the edges, tips and on the surface of interdigitated electrodes. This was the first demonstration of aligned immobilization of proteins by electric fields. Thus, the presented electric field-assisted immobilization method is promising with regard to enhanced antibody binding capacities and enzymatic activities, which is a requirement for industrial biosensor production, as well as for general interaction studies of proteins. N2 - In dieser Doktorarbeit wurde eine Methode entwickelt, mit der Biomoleküle unter dem Einfluss von elektrischen Feldern auf Metallelektroden immobilisiert und ausgerichtet werden können. Für die Immobilisierung wurden weder Modifikationen an den Biomolekülen noch an den Elektroden benötigt. Zwei elektrokinetische Effekte, die Dielektrophorese und der AC-elektroosmotische Fluss, wurden als verantwortliche Effekte für die Molekülbewegung identifiziert. Mit einer neuen 3D Elektrodenkonfiguration wurde der AC-elektroosmotische Fluss minimiert. Damit konnte der Einfluss der experimentellen Parameter auf die Dielektrophoresekraft und deren Auswirkungen auf die Moleküle untersucht werden: Die permanente Immobilisierung von Proteinen wurde mit einem Rasterkraftmikroskop quantifiziert, indem die Volumina der immobilisierten Proteinablagerungen gemessen wurden, und daraus die maximal darin enthaltene Anzahl an Proteinen berechnet wurde. Diese Art der absoluten Quantifizierung war nur möglich, da die Proteine auch nach Abschalten des elektrischen Feldes auf den Wolframelektroden hafteten. Eine solche permanente Immobilisierung funktioneller Proteine auf Elektroden oder Oberflächen im Allgemeinen ist eine wichtige Voraussetzung für die Herstellung von Biosensoren. Des Weiteren muss die Biofunktion der Proteine nach der Immobilisierung erhalten bleiben. Da die Proteine durch die Immobilisierung chemisch oder physikalisch verändert werden, ist auch ihre Biofunktion häufig eingeschränkt. In dieser Arbeit wurde erstmals der Erhalt der Aktivität dielektrophoretisch immobilisierter Enzyme gezeigt. Hierfür wurde das Enzym Meerrettichperoxidase exemplarisch verwendet, dessen Aktivität über die Oxidation von Dihydrorhodamin 123, einem nicht-fluoreszentem Vorläufer des Fluoreszenzfarbstoffes Rhodamin 123, nachgewiesen wurde. Molekulare Ausrichtung und Immobilisierung – sowohl reversibel als auch permanent – wurde unter dem Einfluss inhomogener elektrischer Wechselfelder erreicht. Für die Bestimmung der Molekülausrichtung wurde mit ein Messaufbau entwickelt, der auf einem Fluoreszenzmikroskop basiert. Der Aufbau, das Messprotokoll und die Auswertungsmethode wurden mit einer selbst hergestellten Kontrollprobe, die aus ausgerichteten Acridinorangemolekülen in einem Flüssigkristall bestand, validiert. Lambda-DNA Doppelstränge wurden zwischen benachbarten Interdigitalelektroden gestreckt und temporär ausgerichtet. Die Ausrichtung von interkalierten PicoGreen-Molekülen im rechten Winkel zur Längsachse der Doppelstränge konnte hier gezeigt werden. Zudem konnte die ausgerichtete Immobilisierung des enhanced Green Fluorescent Protein nachgewiesen werden, indem die Fluoreszenz des Proteins und seine Struktureigenschaften ausgenutzt wurden. Aus den Messungen konnte der Winkel des Chromophors relativ zur Proteinlängsachse mit guter Übereinstimmung mit Röntgenkristallstrukturdaten bestimmt werden. Eine permanente Immobilisierung mit gleichzeitiger Ausrichtung der Proteine wurde entlang der Kanten, an den Spitzen und auf der Oberfläche von Interdigitalelektroden erzielt. Damit wurde zum ersten Mal eine ausgerichtete Immobilisierung von Proteinen mit elektrischen Wechselfeldern gezeigt. Diese Methode ist vielversprechend für die Immobilisierung von Antikörpern oder Enzymen mit einheitlicher Ausrichtung und dadurch verbessertem Zugang zu den aktiven Zentren, was nicht nur für die industrielle Biosensorherstellung von Interesse ist, sondern genauso für allgemeine Wechselwirkungsstudien von Proteinen. KW - dielectrophoresis KW - electrokinetics KW - proteins KW - immobilization KW - alignment KW - Dielektrophorese KW - elektrokinetische Effekte KW - Proteine KW - Immobilisierung KW - Ausrichtung Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-90271 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 - 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 - 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 -