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 - 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 - 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 -