TY - JOUR A1 - Kunstmann, Ruth Sonja A1 - Gohlke, Ulrich A1 - Bröker, Nina Kristin A1 - Roske, Yvette A1 - Heinemann, Udo A1 - Santer, Mark A1 - Barbirz, Stefanie T1 - Solvent networks tune thermodynamics of oligosaccharide complex formation in an extended protein binding site JF - Journal of the American Chemical Society N2 - The principles of protein-glycan binding are still not well understood on a molecular level. Attempts to link affinity and specificity of glycan recognition to structure suffer from the general lack of model systems for experimental studies and the difficulty to describe the influence of solvent. We have experimentally and computationally addressed energetic contributions of solvent in protein-glycan complex formation in the tailspike protein (TSP) of E. coli bacteriophage HK620. HK620TSP is a 230 kDa native trimer of right-handed, parallel beta-helices that provide extended, rigid binding sites for bacterial cell surface O-antigen polysaccharides. A set of high affinity mutants bound hexa- or pentasaccharide O-antigen fragments with very similar affinities even though hexasaccharides introduce an additional glucose branch into an occluded protein surface cavity. Remarkably different thermodynamic binding signatures were found for different mutants; however, crystal structure analyses indicated that no major oligosaccharide or protein topology changes had occurred upon complex formation. This pointed to a solvent effect. Molecular dynamics simulations using a mobility-based approach revealed an extended network of solvent positions distributed over the entire oligosaccharide binding site. However, free energy calculations showed that a small water network inside the glucose-binding cavity had the most notable influence on the thermodynamic signature. The energy needed to displace water from the glucose binding pocket depended on the amino acid at the entrance, in agreement with the different amounts of enthalpy-entropy compensation found for introducing glucose into the pocket in the different mutants. Studies with small molecule drugs have shown before that a few active water molecules can control protein complex formation. HK620TSP oligosaccharide binding shows that similar fundamental principles also apply for glycans, where a small number of water molecules can dominate the thermodynamic signature in an extended binding site. Y1 - 2018 U6 - https://doi.org/10.1021/jacs.8b03719 SN - 0002-7863 VL - 140 IS - 33 SP - 10447 EP - 10455 PB - American Chemical Society CY - Washington ER - TY - THES A1 - Kunstmann, Ruth Sonja T1 - Design of a high-affinity carbohydrate binding protein T1 - Design eines hoch-affin Kohlenhydrat-bindenden Proteins N2 - Kohlenhydrat-Protein Interaktionen sind in der Natur weitverbreitet. Sie stellen die Grundlage für viele biologische Prozesse dar, wie zum Beispiel Immunantworten, Wundheilung und Infektionsprozesse von pathogenen Viren oder Bakterien mit einem Wirt wie dem Menschen. Neben der Infektion von Menschen können aber auch Bakterien selbst durch so genannte Bakteriophagen infiziert werden, welche für den Menschen ungefährlich sind. Diese Infektion involviert die spezifische Erkennung der pathogenen Bakterien, die Vermehrung der Bakteriophagen und schließlich die Abtötung der Bakterien. Dabei können die Mechanismen der spezifischen Erkennung genutzt werden, pathogene Bakterien auf Lebensmitteln zu detektieren oder die Diagnose von Infektionen zu vereinfachen. Die spezifische Erkennung von Enteritis-erzeugenden Bakterien wie Escherichia coli, Salmonella spp. oder Shigella flexneri durch Bakteriophagen der Familie der Podoviridae erfolgt über die Bindung eines sogenannten tailspike proteins des Bakteriophagen an das aus Kohlenhydraten-bestehende O-Antigen des Lipopolysaccharids von Gram-negativen Bakterien. Das tailspike protein spaltet das O-Antigen um den Bakteriophage an die Oberfläche des Bakteriums zu führen, damit eine Infektion stattfinden kann. Die Affinität des tailspike proteins zum O-Antigen ist dabei sehr niedrig, um nach Spaltung des O-Antigens das Spaltungsprodukt zu lösen und wiederum neues Substrat zu binden. In dieser Arbeit wurde ein tailspike protein des Bakteriophagen Sf6 verwendet (Sf6 TSP), das spezifisch an das O-Antigen von Shigella flexneri Y bindet. Eine inaktive Variante des Sf6 TSP wurde verwendet um einen hoch-affin bindenden Sensor für pathogene Shigella zu entwickeln. Der Shigella-Sensor wurde durch Kopplung von unterschiedlichen Proteinmutanten mit einem fluoreszierendem Molekül erhalten. Dabei zeigte eine dieser Mutanten bei Bindung von Shigella O-Antigen ein Fluoreszenz-Signal im Bereich des sichtbaren Lichts. Molekulardynamische Simulationen wurde anhand der erzeugten Proteinmutanten als Methode zum rationalen Design von hoch-affin Kohlenhydrat-bindenden Proteinen getestet und die resultierenden Affinitätsvorhersagen wurden über Oberflächenplasmonresonanz-Experimente überprüft. Aus weiteren experimentellen und simulierten Daten konnten schließlich Schlussfolgerungen über die Ursprünge von Kohlenhydrat-Protein Interaktionen gezogen werden, die eine Einsicht über den Einfluss von Wasser in diesem Bindungsprozess lieferten. N2 - Carbohydrate-protein interactions are ubiquitous in nature. They provide the initial molecular contacts in many cell-cell processes as for example immune responses, signal transduction, egg fertilization and infection processes of pathogenic viruses and bacteria. Furthermore, bacteria themselves are infected by bacteriophages, viruses which can cause the bacterial lysis, but do not affect other hosts. The infection process of a bacteriophage involves the specific detection and binding of the bacterium, which can be based on a carbohydrate-protein interaction. The mechanism of specific detection of pathogenic bacteria can thereby be useful for the development of bacteria sensors in the food industry or for tools in diagnostics. Bacteriophages of the Podoviridae family use tailspike proteins for the specific detection of enteritis causing bacteria as Escherichia coli, Salmonella spp. or Shigella flexneri. The tailspike protein provides the first contact by binding to the carbohydrate containing O-antigen part of lipopolysaccharide in the Gram-negative cell wall. After binding to O-antigen repeating units, the enzymatic activity of tailspike proteins leads to cleavage of the carbohydrate chains, which enables the bacteriophage to approach the bacterial surface for DNA injection. Tailspike proteins thereby exhibit a relatively low affinity to the oligosaccharide structures of O-antigen due to the necessary binding, cleavage and release cycle, compared for example to antibodies. In this work it was aimed to study the determinants that influence carbohydrate affinity in the extended TSP binding grooves. This is a prerequisite to design a high-affinity tailspike protein based bacteria sensor. For this purpose the tailspike protein of the bacteriophage Sf6 (Sf6 TSP) was used, which specifically binds Shigella flexneri Y O-antigen with two tetrasaccharide repeating units at the intersubunits of the trimeric β-helix protein. The Sf6 TSP endorhamnosidase cleaves the O-antigen, which leads to an octasaccharide as the main product. The binding affinity of inactive Sf6 TSP towards polysaccharide was characterized by fluorescence titration experiments and surface plasmon resonance (SPR). Moreover, cysteine mutations were introduced into the Sf6 TSP binding site for the covalent thiol-coupling of an environment-sensitive fluorescent label to obtain a sensor for Shigella flexneri Y based on TSP-O-antigen recognition. This sensor showed a more than 100 % amplitude increase of a visible light fluorescence upon the binding of a polysaccharide test solution. Improvements of the TSP sensor can be achieved by increasing the tailspike affinity towards the O-antigen. Therefore molecular dynamics simulations evaluating ligand flexibility, hydrogen bond occupancies and water network distributions were used for affinity prediction on the available cysteine mutants of Sf6 TSP. The binding affinities were experimentally analyzed by SPR. This combined computational and experimental set-up for the design of a high-affinity carbohydrate binding protein could successfully distinguish strongly increased and decreased affinities of single amino acid mutants. A thermodynamically and structurally well characterized set of another tailspike protein HK620 TSP with high-affinity mutants was used to evaluate the influence of water molecules on binding affinity. The free enthalpy of HK620 TSP oligosaccharide complex formation thereby either derived from the replacement of a conserved water molecule or by immobilization of two water molecules upon ligand binding. Furthermore, the enthalpic and entropic contributions of water molecules in a hydrophobic binding pocket could be assigned by free energy calculations. The findings in this work can be helpful for the improvement of carbohydrate docking and carbohydrate binding protein engineering algorithms in the future. KW - Kohlenhydrat-Protein Interaction KW - carbohydrate-protein interaction KW - bacterial sensor KW - Bakterien Sensor Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-403458 ER - TY - GEN A1 - Kunstmann, Ruth Sonja A1 - Scheidt, Tom A1 - Buchwald, Saskia A1 - Helm, Alexandra A1 - Mulard, Laurence A. A1 - Fruth, Angelika A1 - Barbirz, Stefanie T1 - Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens T2 - Viruses N2 - Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 472 KW - Shigella flexneri KW - bacteriophage KW - tailspike proteins KW - O-antigen KW - serotyping KW - microtiter plate assay KW - fluorescence sensor Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-417831 ER - TY - JOUR A1 - Kunstmann, Ruth Sonja A1 - Scheidt, Tom A1 - Buchwald, Saskia A1 - Helm, Alexandra A1 - Mulard, Laurence A. A1 - Fruth, Angelika A1 - Barbirz, Stefanie T1 - Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens JF - Viruses N2 - Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system. KW - Shigella flexneri KW - bacteriophage KW - tailspike proteins KW - O-antigen KW - serotyping KW - microtiter plate assay KW - fluorescence sensor Y1 - 2018 U6 - https://doi.org/10.3390/v10080431 SN - 1999-4915 VL - 10 IS - 8 SP - 1 EP - 18 PB - Molecular Diversity Preservation International (MDPI) CY - Basel ER - TY - GEN A1 - Georgiev, Vasil N. A1 - Grafmüller, Andrea A1 - Bléger, David A1 - Hecht, Stefan A1 - Kunstmann, Ruth Sonja A1 - Barbirz, Stefanie A1 - Lipowsky, Reinhard A1 - Dimova, Rumiana T1 - Area increase and budding in giant vesicles triggered by light BT - behind the scene T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Biomembranes are constantly remodeled and in cells, these processes are controlled and modulated by an assortment of membrane proteins. Here, it is shown that such remodeling can also be induced by photoresponsive molecules. The morphological control of giant vesicles in the presence of a water-soluble ortho-tetrafluoroazobenzene photoswitch (F-azo) is demonstrated and it is shown that the shape transformations are based on an increase in membrane area and generation of spontaneous curvature. The vesicles exhibit budding and the buds can be retracted by using light of a different wavelength. In the presence of F-azo, the membrane area can increase by more than 5% as assessed from vesicle electrodeformation. To elucidate the underlying molecular mechanism and the partitioning of F-azo in the membrane, molecular dynamics simulations are employed. Comparison with theoretically calculated shapes reveals that the budded shapes are governed by curvature elasticity, that the spontaneous curvature can be decomposed into a local and a nonlocal contribution, and that the local spontaneous curvature is about 1/(2.5 mu m). The results show that exo- and endocytotic events can be controlled by light and that these photoinduced processes provide an attractive method to change membrane area and morphology. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 733 KW - azobenzene KW - lipid membranes KW - molecular dynamics KW - photoswitch KW - vesicles Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-426298 SN - 1866-8372 VL - 5 IS - 733 ER - TY - JOUR A1 - Kang, Yu A1 - Gohlke, Ulrich A1 - Engström, Olof A1 - Hamark, Christoffer A1 - Scheidt, Tom A1 - Kunstmann, Ruth Sonja A1 - Heinemann, Udo A1 - Widmalm, Göran A1 - Santer, Mark A1 - Barbirz, Stefanie T1 - Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions JF - Journal of the American Chemical Society N2 - Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system. Y1 - 2016 U6 - https://doi.org/10.1021/jacs.6b00240 SN - 0002-7863 VL - 138 SP - 9109 EP - 9118 PB - American Chemical Society CY - Washington ER - TY - GEN A1 - Kunstmann, Ruth Sonja A1 - Engström, Olof A1 - Wehle, Marko A1 - Widmalm, Göran A1 - Santer, Mark A1 - Barbirz, Stefanie T1 - Increasing the affinity of an O-Antigen polysaccharide binding site in Shigella flexneri bacteriophage Sf6 tailspike protein T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen-binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O-polysaccharide of dysentery-causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D H-1,H-1-trNOESY NMR utilizing methine-methine and methine-methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O-polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP-based pathogen sensor design. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1417 KW - carbohydrates KW - molecular dynamics simulations KW - NMR spectroscopy KW - protein-carbohydrate interactions KW - surface plasmon resonance Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-519418 SN - 1866-8372 IS - 32 ER - TY - JOUR A1 - Kunstmann, Ruth Sonja A1 - Engström, Olof A1 - Wehle, Marko A1 - Widmalm, Göran A1 - Santer, Mark A1 - Barbirz, Stefanie T1 - Increasing the affinity of an O-Antigen polysaccharide binding site in Shigella flexneri bacteriophage Sf6 tailspike protein JF - Chemistry – A European Journal N2 - Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen-binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O-polysaccharide of dysentery-causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D H-1,H-1-trNOESY NMR utilizing methine-methine and methine-methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O-polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP-based pathogen sensor design. KW - carbohydrates KW - molecular dynamics simulations KW - NMR spectroscopy KW - protein-carbohydrate interactions KW - surface plasmon resonance Y1 - 2020 U6 - https://doi.org/10.1002/chem.202000495 SN - 0947-6539 SN - 1521-3765 VL - 26 IS - 32 SP - 7263 EP - 7273 PB - Wiley-VCH CY - Weinheim ER -