@article{ZhangChenSiemiatkowskaetal.2020, author = {Zhang, Youjun and Chen, Moxian and Siemiatkowska, Beata and Toleco, Mitchell Rey and Jing, Yue and Strotmann, Vivien and Zhang, Jianghua and Stahl, Yvonne and Fernie, Alisdair R.}, title = {A highly efficient agrobacterium-mediated method for transient gene expression and functional studies in multiple plant species}, series = {Plant Communications}, volume = {1}, journal = {Plant Communications}, number = {5}, publisher = {Science Direct}, address = {New York}, issn = {2590-3462}, pages = {12}, year = {2020}, abstract = {Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.}, language = {en} } @misc{ZhangChenSiemiatkowskaetal.2020, author = {Zhang, Youjun and Chen, Moxian and Siemiatkowska, Beata and Toleco, Mitchell Rey and Jing, Yue and Strotmann, Vivien and Zhang, Jianghua and Stahl, Yvonne and Fernie, Alisdair R.}, title = {A highly efficient agrobacterium-mediated method for transient gene expression and functional studies in multiple plant species}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {5}, issn = {1866-8372}, doi = {10.25932/publishup-52425}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-524254}, pages = {14}, year = {2020}, abstract = {Although the use of stable transformation technology has led to great insight into gene function, its application in high-throughput studies remains arduous. Agro-infiltration have been widely used in species such as Nicotiana benthamiana for the rapid detection of gene expression and protein interaction analysis, but this technique does not work efficiently in other plant species, including Arabidopsis thaliana. As an efficient high-throughput transient expression system is currently lacking in the model plant species A. thaliana, we developed a method that is characterized by high efficiency, reproducibility, and suitability for transient expression of a variety of functional proteins in A. thaliana and 7 other plant species, including Brassica oleracea, Capsella rubella, Thellungiella salsuginea, Thellungiella halophila, Solanum tuberosum, Capsicum annuum, and N. benthamiana. Efficiency of this method was independently verified in three independent research facilities, pointing to the robustness of this technique. Furthermore, in addition to demonstrating the utility of this technique in a range of species, we also present a case study employing this method to assess protein-protein interactions in the sucrose biosynthesis pathway in Arabidopsis.}, language = {en} } @phdthesis{Paul2017, author = {Paul, Fabian}, title = {Markov state modeling of binding and conformational changes of proteins}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-404273}, school = {Universit{\"a}t Potsdam}, pages = {X, 112}, year = {2017}, abstract = {Proteins are molecules that are essential for life and carry out an enormous number of functions in organisms. To this end, they change their conformation and bind to other molecules. However, the interplay between conformational change and binding is not fully understood. In this work, this interplay is investigated with molecular dynamics (MD) simulations of the protein-peptide system Mdm2-PMI and by analysis of data from relaxation experiments. The central task it to uncover the binding mechanism, which is described by the sequence of (partial) binding events and conformational change events including their probabilities. In the simplest case, the binding mechanism is described by a two-step model: binding followed by conformational change or conformational change followed by binding. In the general case, longer sequences with multiple conformational changes and partial binding events are possible as well as parallel pathways that differ in their sequences of events. The theory of Markov state models (MSMs) provides the theoretical framework in which all these cases can be modeled. For this purpose, MSMs are estimated in this work from MD data, and rate equation models, which are related to MSMs, are inferred from experimental relaxation data. The MD simulation and Markov modeling of the PMI-Mdm2 system shows that PMI and Mdm2 can bind via multiple pathways. A main result of this work is a dissociation rate on the order of one event per second, which was calculated using Markov modeling and is in agreement with experiment. So far, dissociation rates and transition rates of this magnitude have only been calculated with methods that speed up transitions by acting with time-dependent, external forces on the binding partners. The simulation technique developed in this work, in contrast, allows the estimation of dissociation rates from the combination of free energy calculation and direct MD simulation of the fast binding process. Two new statistical estimators TRAM and TRAMMBAR are developed to estimate a MSM from the joint data of both simulation types. In addition, a new analysis technique for time-series data from chemical relaxation experiments is developed in this work. It allows to identify one of the above-mentioned two-step mechanisms as the mechanism that underlays the data. The new method is valid for a broader range of concentrations than previous methods and therefore allows to choose the concentrations such that the mechanism can be uniquely identified. It is successfully tested with data for the binding of recoverin to a rhodopsin kinase peptide.}, language = {en} } @article{NietzscheSchiesslBoernke2014, author = {Nietzsche, Madlen and Schiessl, Ingrid and B{\"o}rnke, Frederik}, title = {The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell and stimulus type-specific SnRK1 signaling in plants}, series = {Frontiers in plant science}, volume = {5}, journal = {Frontiers in plant science}, publisher = {Frontiers Research Foundation}, address = {Lausanne}, issn = {1664-462X}, doi = {10.3389/fpls.2014.00054}, pages = {13}, year = {2014}, abstract = {In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic alpha subunit associates with a regulatory beta subunit and an activating gamma subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1 alpha subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.}, language = {en} } @article{HagenMattayRaeuberetal.2014, author = {Hagen, Sven and Mattay, Dinah and Raeuber, Christina and Mueller, Kristian M. and Arndt, Katja Maren}, title = {Characterization and inhibition of AF10-mediated interaction}, series = {Journal of peptide science}, volume = {20}, journal = {Journal of peptide science}, number = {6}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1075-2617}, doi = {10.1002/psc.2626}, pages = {385 -- 397}, year = {2014}, abstract = {The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14-21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leukemogenic effect, has been shown to interact with GAS41, a protein previously identified as the product of an amplified gene in glioblastoma. Using sequential synthetic peptides, we mapped the potential AF10/GAS41 interaction site, which was subsequently be used as scaffold for a library targeting the AF10 coiled-coil domain. Using phage display, we selected a peptide that binds the AF10 coiled-coil domain with higher affinity than the respective coiled-coil region of wild-type GAS41, as demonstrated by phage ELISA, CD, and PCAs. Furthermore, we were able to successfully deploy the inhibitory peptide in a mammalian cell line to lower the expression of Hoxa genes that have been described to be overexpressed in these leukemias. This work dissects molecular determinants mediating AF10-directed interactions in leukemic fusions comprising the N-terminal parts of the proteins MLL or CALM and the C-terminal coiled-coil domain of AF10. Furthermore, it outlines the first steps in recognizing and blocking the leukemia-associated AF10 interaction in histiocytic lymphoma cells and therefore, may have significant implications in future diagnostics and therapeutics. Copyright (c) 2014 European Peptide Society and John Wiley \& Sons, Ltd.}, language = {en} } @article{RaufArifDortayetal.2013, author = {Rauf, Mamoona and Arif, Muhammad and Dortay, Hakan and Matallana-Ramirez, Lilian P. and Waters, Mark T. and Nam, Hong Gil and Lim, Pyung-Ok and M{\"u}ller-R{\"o}ber, Bernd and Balazadeh, Salma}, title = {ORE1 balances leaf senescence against maintenance by antagonizing G2-like-mediated transcription}, series = {EMBO reports}, volume = {14}, journal = {EMBO reports}, number = {4}, publisher = {Nature Publ. Group}, address = {London}, issn = {1469-221X}, doi = {10.1038/embor.2013.24}, pages = {382 -- 388}, year = {2013}, abstract = {Leaf senescence is a key physiological process in all plants. Its onset is tightly controlled by transcription factors, of which NAC factor ORE1 (ANAC092) is crucial in Arabidopsis thaliana. Enhanced expression of ORE1 triggers early senescence by controlling a downstream gene network that includes various senescence-associated genes. Here, we report that unexpectedly ORE1 interacts with the G2-like transcription factors GLK1 and GLK2, which are important for chloroplast development and maintenance, and thereby for leaf maintenance. ORE1 antagonizes GLK transcriptional activity, shifting the balance from chloroplast maintenance towards deterioration. Our finding identifies a new mechanism important for the control of senescence by ORE1.}, language = {en} }