@phdthesis{Schlossarek2023, author = {Schlossarek, Dennis}, title = {Identification of dynamic protein-metabolite complexes in saccharomyces cerevisiae using co-fractionation mass spectrometry}, doi = {10.25932/publishup-58282}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-582826}, school = {Universit{\"a}t Potsdam}, pages = {123}, year = {2023}, abstract = {Cells are built from a variety of macromolecules and metabolites. Both, the proteome and the metabolome are highly dynamic and responsive to environmental cues and developmental processes. But it is not their bare numbers, but their interactions that enable life. The protein-protein (PPI) and protein-metabolite interactions (PMI) facilitate and regulate all aspects of cell biology, from metabolism to mitosis. Therefore, the study of PPIs and PMIs and their dynamics in a cell-wide context is of great scientific interest. In this dissertation, I aim to chart a map of the dynamic PPIs and PMIs across metabolic and cellular transitions. As a model system, I study the shift from the fermentative to the respiratory growth, known as the diauxic shift, in the budding yeast Saccharomyces cerevisiae. To do so, I am applying a co-fractionation mass spectrometry (CF-MS) based method, dubbed protein metabolite interactions using size separation (PROMIS). PROMIS, as well as comparable methods, will be discussed in detail in chapter 1. Since PROMIS was developed originally for Arabidopsis thaliana, in chapter 2, I will describe the adaptation of PROMIS to S. cerevisiae. Here, the obtained results demonstrated a wealth of protein-metabolite interactions, and experimentally validated 225 previously predicted PMIs. Applying orthogonal, targeted approaches to validate the interactions of a proteogenic dipeptide, Ser-Leu, five novel protein-interactors were found. One of those proteins, phosphoglycerate kinase, is inhibited by Ser-Leu, placing the dipeptide at the regulation of glycolysis. In chapter 3, I am presenting PROMISed, a novel web-tool designed for the analysis of PROMIS- and other CF-MS-datasets. Starting with raw fractionation profiles, PROMISed enables data pre-processing, profile deconvolution, scores differences in fractionation profiles between experimental conditions, and ultimately charts interaction networks. PROMISed comes with a user-friendly graphic interface, and thus enables the routine analysis of CF-MS data by non-computational biologists. Finally, in chapter 4, I applied PROMIS in combination with the isothermal shift assay to the diauxic shift in S. cerevisiae to study changes in the PPI and PMI landscape across this metabolic transition. I found a major rewiring of protein-protein-metabolite complexes, exemplified by the disassembly of the proteasome in the respiratory phase, the loss of interaction of an enzyme involved in amino acid biosynthesis and its cofactor, as well as phase and structure specific interactions between dipeptides and enzymes of central carbon metabolism. In chapter 5, I am summarizing the presented results, and discuss a strategy to unravel the potential patterns of dipeptide accumulation and binding specificities. Lastly, I recapitulate recently postulated guidelines for CF-MS experiments, and give an outlook of protein interaction studies in the near future.}, language = {en} } @article{EilersKleineEckertetal.2021, author = {Eilers, Elisabeth Johanna and Kleine, Sandra and Eckert, Silvia and Waldherr, Simon and M{\"u}ller, Caroline}, title = {Flower production, headspace volatiles, pollen nutrients, and florivory in tanacetum vulgare chemotypes}, series = {Frontiers in plant science : FPLS}, volume = {11}, journal = {Frontiers in plant science : FPLS}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {1664-462X}, doi = {10.3389/fpls.2020.611877}, pages = {17}, year = {2021}, abstract = {Floral volatiles and reward traits are major drivers for the behavior of mutualistic as well as antagonistic flower visitors, i.e., pollinators and florivores. These floral traits differ tremendously between species, but intraspecific differences and their consequences on organism interactions remain largely unknown. Floral volatile compounds, such as terpenoids, function as cues to advertise rewards to pollinators, but should at the same time also repel florivores. The reward composition, e.g., protein and lipid contents in pollen, differs between individuals of distinct plant families. Whether the nutritional value of rewards within the same plant species is linked to their chemotypes, which differ in their pattern of specialized metabolites, has yet not been investigated. In the present study, we compared Tanacetum vulgare plants of five terpenoid chemotypes with regard to flower production, floral headspace volatiles, pollen macronutrient and terpenoid content, and floral attractiveness to florivorous beetles. Our analyses revealed remarkable differences between the chemotypes in the amount and diameter of flower heads, duration of bloom period, and pollen nutritional quality. The floral headspace composition of pollen-producing mature flowers, but not of premature flowers, was correlated to that of pollen and leaves in the same plant individual. For two chemotypes, florivorous beetles discriminated between the scent of mature and premature flower heads and preferred the latter. In semi-field experiments, the abundance of florivorous beetles and flower tissue miners differed between T. vulgare chemotypes. Moreover, the scent environment affected the choice and beetles were more abundant in homogenous plots composed of one single chemotype than in plots with different neighboring chemotypes. In conclusion, flower production, floral metabolic composition and pollen quality varied to a remarkable extend within the species T. vulgare, and the attractiveness of floral scent differed also intra-individually with floral ontogeny. We found evidence for a trade-off between pollen lipid content and pollen amount on a per-plant-level. Our study highlights that chemotypes which are more susceptible to florivory are less attacked when they grow in the neighborhood of other chemotypes and thus gain a benefit from high overall chemodiversity.}, language = {en} } @phdthesis{Banerjee2020, author = {Banerjee, Pallavi}, title = {Glycosylphosphatidylinositols (GPIs) and GPI-anchored proteins tethered to lipid bilayers}, doi = {10.25932/publishup-48956}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-489561}, school = {Universit{\"a}t Potsdam}, pages = {xv, 141}, year = {2020}, abstract = {Glycosylphosphatidylinositols (GPIs) are highly complex glycolipids that serve as membrane anchors to a large variety of eukaryotic proteins. These are covalently attached to a group of peripheral proteins called GPI-anchored proteins (GPI-APs) through a post-translational modification in the endoplasmic reticulum. The GPI anchor is a unique structure composed of a glycan, with phospholipid tail at one end and a phosphoethanolamine linker at the other where the protein attaches. The glycan part of the GPI comprises a conserved pseudopentasaccharide core that could branch out to carry additional glycosyl or phosphoethanolamine units. GPI-APs are involved in a diverse range of cellular processes, few of which are signal transduction, protein trafficking, pathogenesis by protozoan parasites like the malaria- causing parasite Plasmodium falciparum. GPIs can also exist freely on the membrane surface without an attached protein such as those found in parasites like Toxoplasma gondii, the causative agent of Toxoplasmosis. These molecules are both structurally and functionally diverse, however, their structure-function relationship is still poorly understood. This is mainly because no clear picture exists regarding how the protein and the glycan arrange with respect to the lipid layer. Direct experimental evidence is rather scarce, due to which inconclusive pictures have emerged, especially regarding the orientation of GPIs and GPI-APs on membrane surfaces and the role of GPIs in membrane organization. It appears that computational modelling through molecular dynamics simulations would be a useful method to make progress. In this thesis, we attempt to explore characteristics of GPI anchors and GPI-APs embedded in lipid bilayers by constructing molecular models at two different resolutions - all-atom and coarse-grained. First, we show how to construct a modular molecular model of GPIs and GPI-anchored proteins that can be readily extended to a broad variety of systems, addressing the micro-heterogeneity of GPIs. We do so by creating a hybrid link to which GPIs of diverse branching and lipid tails of varying saturation with their optimized force fields, GLYCAM06 and Lipid14 respectively, can be attached. Using microsecond simulations, we demonstrate that GPI prefers to "flop-down" on the membrane, thereby, strongly interacting with the lipid heads, over standing upright like a "lollipop". Secondly, we extend the model of the GPI core to carry out a systematic study of the structural aspects of GPIs carrying different side chains (parasitic and human GPI variants) inserted in lipid bilayers. Our results demonstrate the importance of the side branch residues as these are the most accessible, and thereby, recognizable epitopes. This finding qualitatively agrees with experimental observations that highlight the role of the side branches in immunogenicity of GPIs and the specificity thereof. The overall flop-down orientation of the GPIs with respect to the bilayer surface presents the side chain residues to face the solvent. Upon attaching the green fluorescent protein (GFP) to the GPI, it is seen to lie in close proximity to the bilayer, interacting both with the lipid heads and glycan part of the GPI. However the orientation of GFP is sensitive to the type of GPI it is attached to. Finally, we construct a coarse-grained model of the GPI and GPI-anchored GFP using a modified version of the MARTINI force-field, using which the timescale is enhanced by at least an order of magnitude compared to the atomistic system. This study provides a theoretical perspective on the conformational behavior of the GPI core and some of its branched variations in presence of lipid bilayers, as well as draws comparisons with experimental observations. Our modular atomistic model of GPI can be further employed to study GPIs of variable branching, and thereby, aid in designing future experiments especially in the area of vaccines and drug therapies. Our coarse-grained model can be used to study dynamic aspects of GPIs and GPI-APs w.r.t plasma membrane organization. Furthermore, the backmapping technique of converting coarse-grained trajectory back to the atomistic model would enable in-depth structural analysis with ample conformational sampling.}, language = {en} } @misc{BiterovaEsmaeeliMoghaddamTabalvandaniAlanenetal.2018, author = {Biterova, Ekaterina and Esmaeeli Moghaddam Tabalvandani, Mariam and Alanen, Heli I. and Saaranen, Mirva and Ruddock, Lloyd W.}, title = {Structures of Angptl3 and Angptl4}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1048}, issn = {1866-8372}, doi = {10.25932/publishup-46794}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-467943}, pages = {14}, year = {2018}, abstract = {Coronary artery disease is the most common cause of death globally and is linked to a number of risk factors including serum low density lipoprotein, high density lipoprotein, triglycerides and lipoprotein(a). Recently two proteins, angiopoietin-like protein 3 and 4, have emerged from genetic studies as being factors that significantly modulate plasma triglyceride levels and coronary artery disease. The exact function and mechanism of action of both proteins remains to be elucidated, however, mutations in these proteins results in up to 34\% reduction in coronary artery disease and inhibition of function results in reduced plasma triglyceride levels. Here we report the crystal structures of the fibrinogen-like domains of both proteins. These structures offer new insights into the reported loss of function mutations, the mechanisms of action of the proteins and open up the possibility for the rational design of low molecular weight inhibitors for intervention in coronary artery disease.}, language = {en} } @misc{KuekenSommerYanevaRoderetal.2018, author = {K{\"u}ken, Anika and Sommer, Frederik and Yaneva-Roder, Liliya and Mackinder, Luke C.M. and H{\"o}hne, Melanie and Geimer, Stefan and Jonikas, Martin C. and Schroda, Michael and Stitt, Mark and Nikoloski, Zoran and Mettler-Altmann, Tabea}, title = {Effects of microcompartmentation on flux distribution and metabolic pools in Chlamydomonas reinhardtii chloroplasts}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1122}, issn = {1866-8372}, doi = {10.25932/publishup-44635}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-446358}, pages = {25}, year = {2018}, abstract = {Cells and organelles are not homogeneous but include microcompartments that alter the spatiotemporal characteristics of cellular processes. The effects of microcompartmentation on metabolic pathways are however difficult to study experimentally. The pyrenoid is a microcompartment that is essential for a carbon concentrating mechanism (CCM) that improves the photosynthetic performance of eukaryotic algae. Using Chlamydomonas reinhardtii, we obtained experimental data on photosynthesis, metabolites, and proteins in CCM-induced and CCM-suppressed cells. We then employed a computational strategy to estimate how fluxes through the Calvin-Benson cycle are compartmented between the pyrenoid and the stroma. Our model predicts that ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco, and 3-phosphoglycerate (3PGA), its product, diffuse in and out of the pyrenoid, respectively, with higher fluxes in CCM-induced cells. It also indicates that there is no major diffusional barrier to metabolic flux between the pyrenoid and stroma. Our computational approach represents a stepping stone to understanding microcompartmentalized CCM in other organisms.}, language = {en} } @misc{LukanMachensColletal.2018, author = {Lukan, Tjaša and Machens, Fabian and Coll, Anna and Baebler, Špela and Messerschmidt, Katrin and Gruden, Kristina}, title = {Plant X-tender}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {990}, issn = {1866-8372}, doi = {10.25932/publishup-44628}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-446281}, pages = {21}, year = {2018}, abstract = {Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scarfree and sequence-independent multigene assembly strategy AssemblX,based on overlapdepended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.}, language = {en} } @article{LauxErmilovaPannwitzetal.2018, author = {Laux, Eva-Maria and Ermilova, Elena and Pannwitz, Daniel and Gibbons, Jessica and H{\"o}lzel, Ralph and Bier, Frank Fabian}, title = {Dielectric Spectroscopy of Biomolecules up to 110 GHz}, series = {Frequenz}, volume = {72}, journal = {Frequenz}, number = {3-4}, publisher = {De Gruyter}, address = {Berlin}, issn = {0016-1136}, doi = {10.1515/freq-2018-0010}, pages = {135 -- 140}, year = {2018}, abstract = {Radio-frequency fields in the GHz range are increasingly applied in biotechnology and medicine. In order to fully exploit both their potential and their risks detailed information about the dielectric properties of biological material is needed. For this purpose a measuring system is presented that allows the acquisition of complex dielectric spectra over 4 frequency decade up to 110 GHz. Routines for calibration and for data evaluation according to physicochemical interaction models have been developed. The frequency dependent permittivity and dielectric loss of some proteins and nucleic acids, the main classes of biomolecules, and of their sub-units have been determined. Dielectric spectra are presented for the amino acid alanine, the proteins lysozyme and haemoglobin, the nucleotides AMP and ATP, and for the plasmid pET-21, which has been produced by bacterial culture. Characterisation of a variety of biomolecules is envisaged, as is the application to studies on protein structure and function.}, language = {en} } @article{LaemkeBaeurle2017, author = {L{\"a}mke, J{\"o}rn and B{\"a}urle, Isabel}, title = {Epigenetic and chromatin-based mechanisms in environmental stress adaptation and stress memory in plants}, series = {Genome biology : biology for the post-genomic era}, volume = {18}, journal = {Genome biology : biology for the post-genomic era}, publisher = {BioMed Central}, address = {London}, issn = {1474-760X}, doi = {10.1186/s13059-017-1263-6}, pages = {8685 -- 8693}, year = {2017}, abstract = {Plants frequently have to weather both biotic and abiotic stressors, and have evolved sophisticated adaptation and defense mechanisms. In recent years, chromatin modifications, nucleosome positioning, and DNA methylation have been recognized as important components in these adaptations. Given their potential epigenetic nature, such modifications may provide a mechanistic basis for a stress memory, enabling plants to respond more efficiently to recurring stress or even to prepare their offspring for potential future assaults. In this review, we discuss both the involvement of chromatin in stress responses and the current evidence on somatic, intergenerational, and transgenerational stress memory.}, language = {en} } @misc{WessigBaderKlieretal.2016, author = {Wessig, Pablo and Bader, Denise and Klier, Dennis Tobias and Hettrich, Cornelia and Bier, Frank Fabian}, title = {Detecting carbohydrate-lectin interactions using a fluorescent probe based on DBD dyes}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-394382}, pages = {1235 -- 1238}, year = {2016}, abstract = {Herein we present an efficient synthesis of a biomimetic probe with modular construction that can be specifically bound by the mannose binding FimH protein - a surface adhesion protein of E. coli bacteria. The synthesis combines the new and interesting DBD dye with the carbohydrate ligand mannose via a Click reaction. We demonstrate the binding to E. coli bacteria over a large concentration range and also present some special characteristics of those molecules that are of particular interest for the application as a biosensor. In particular, the mix-and-measure ability and the very good photo-stability should be highlighted here.}, language = {en} } @phdthesis{Wettstein2015, author = {Wettstein, Christoph}, title = {Cytochrome c-DNA and cytochrome c-enzyme interactions for the construction of analytical signal chains}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-78367}, school = {Universit{\"a}t Potsdam}, pages = {120}, year = {2015}, abstract = {Electron transfer (ET) reactions play a crucial role in the metabolic pathways of all organisms. In biotechnological approaches, the redox properties of the protein cytochrome c (cyt c), which acts as an electron shuttle in the respiratory chain, was utilized to engineer ET chains on electrode surfaces. With the help of the biopolymer DNA, the redox protein assembles into electro active multilayer (ML) systems, providing a biocompatible matrix for the entrapment of proteins. In this study the characteristics of the cyt c and DNA interaction were defined on the molecular level for the first time and the binding sites of DNA on cyt c were identified. Persistent cyt c/DNA complexes were formed in solution under the assembly conditions of ML architectures, i.e. pH 5.0 and low ionic strength. At pH 7.0, no agglomerates were formed, permitting the characterization of the NMR spectroscopy. Using transverse relaxation-optimized spectroscopy (TROSY)-heteronuclear single quantum coherence (HSQC) experiments, DNAs' binding sites on the protein were identified. In particular, negatively charged AA residues, which are known interaction sites in cyt c/protein binding were identified as the main contact points of cyt c and DNA. Moreover, the sophisticated task of arranging proteins on electrode surfaces to create functional ET chains was addressed. Therefore, two different enzyme types, the flavin dependent fructose dehydrogenase (FDH) and the pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH), were tested as reaction partners of freely diffusing cyt c and cyt c immobilized on electrodes in mono- and MLs. The characterisation of the ET processes was performed by means of electrochemistry and the protein deposition was monitored by microgravimetric measurements. FDH and PQQ-GDH were found to be generally suitable for combination with the cyt c/DNA ML system, since both enzymes interact with cyt c in solution and in the immobilized state. The immobilization of FDH and cyt c was achieved with the enzyme on top of a cyt c monolayer electrode without the help of a polyelectrolyte. Combining FDH with the cyt c/DNA ML system did not succeed, yet. However, the basic conditions for this protein-protein interaction were defined. PQQ-GDH was successfully coupled with the ML system, demonstrating that that the cyt c/DNA ML system provides a suitable interface for enzymes and that the creation of signal chains, based on the idea of co-immobilized proteins is feasible. Future work may be directed to the investigation of cyt c/DNA interaction under the precise conditions of ML assembly. Therefore, solid state NMR or X-ray crystallography may be required. Based on the results of this study, the combination of FDH with the ML system should be addressed. Moreover, alternative types of enzymes may be tested as catalytic component of the ML assembly, aiming on the development of innovative biosensor applications.}, language = {en} } @phdthesis{Lengefeld2010, author = {Lengefeld, Jan}, title = {Zirkulardichroismus-Messungen mit Synchrotronstrahlung am BESSY : M{\"o}glichkeiten und Grenzen bei der Untersuchung biologischer Proben}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-44263}, school = {Universit{\"a}t Potsdam}, year = {2010}, abstract = {In dieser Arbeit wurden die M{\"o}glichkeiten und Grenzen f{\"u}r Zirkulardichroismus-Messungen mit Synchrotronstrahlung untersucht. Dazu wurde ein Messaufbau f{\"u}r Zirkulardichroismus-Messungen an zwei Strahlrohren am Berliner Elektronenspeicherring f{\"u}r Synchrotronstrahlung eingesetzt, die f{\"u}r Messungen im Bereich des ultravioletten Lichts geeignet sind. Eigenschaften der Strahlrohre und des Messaufbau wurden in einigen wichtigen Punkten mit kommerziellen Zirkulardichroismus-Spektrometern verglichen. Der Schwerpunkt lag auf der Ausdehnung des zug{\"a}nglichen Wellenl{\"a}ngenbereichs unterhalb von 180 nm zur Untersuchung des Zirkulardichroismus von Proteinen in diesem Bereich. In diesem Bereich ist es nicht nur die Lichtquelle sondern vor allem die Absorption des Lichts durch Wasser, die den Messbereich bei der Messung biologischer Proben in w{\"a}ssriger L{\"o}sung einschr{\"a}nkt. Es wurden Bedingungen gefunden, unter denen der Messbereich auf etwa 160 nm, in einigen F{\"a}llen bis auf 130 nm ausgedehnt werden konnte. Dazu musste die Pfadl{\"a}nge deutlich reduziert werden und verschieden Probenk{\"u}vetten wurden getestet. Der Einfluss der dabei auftretenden Spannungsdoppelbrechung in den Probenk{\"u}vetten auf das Messsignal konnte mit einem alternativen Messaufbau deutlich reduziert werden. Systematische Fehler im Messsignal und auftretende Strahlensch{\"a}den begrenzen jedoch die Zuverl{\"a}ssigkeit der gemessenen Spektren. Bei Proteinfilmen schr{\"a}nkt die Absorption von Wasser den Messbereich kaum ein. Es wurden jedoch meist deutliche Unterschiede zwischen den Spektren von Proteinfilmen und den Spektren von Proteinen in w{\"a}ssriger L{\"o}sung festgestellt. Solange diese Unterschiede nicht minimiert werden k{\"o}nnen, stellen Proteinfilme keine praktikable Alternative zu Messungen in w{\"a}ssriger L{\"o}sung dar.}, language = {de} }