Biaffin GmbH and Co. KG

Kassel, Germany

Biaffin GmbH and Co. KG

Kassel, Germany
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Knape M.J.,University of Kassel | Ahuja L.G.,University of California at San Diego | Bertinetti D.,University of Kassel | Burghardt N.C.G.,University of Kassel | And 3 more authors.
ACS Chemical Biology | Year: 2015

cAMP-dependent protein kinase (PKA) is regulated primarily in response to physiological signals while nucleotides and metals may provide fine-tuning. PKA can use different metal ions for phosphoryl transfer, yet some, like Ca2+, do not support steady-state catalysis. Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR) were used to study inhibitor and substrate interactions with PKA. The data illustrate how metals can act differentially as a result of their inherent coordination properties. We found that Ca2+, in contrast to Mg2+, does not induce high-affinity binding of PKA to pseudosubstrate inhibitors. However, Ca2+ works in a single turnover mode to allow for phosphoryl-transfer. Using a novel SPR approach, we were able to directly monitor the interaction of PKA with a substrate in the presence of Mg2+ATP. This allows us to depict the entire kinase reaction including complex formation as well as release of the phosphorylated substrate. In contrast to Mg2+, Ca2+ apparently slows down the enzymatic reaction. A focus on individual reaction steps revealed that Ca2+ is not as efficient as Mg2+ in stabilizing the enzyme:substrate complex. The opposite holds true for product dissociation where Mg2+ easily releases the phospho-substrate while Ca2+ traps both reaction products at the active site. This explains the low steady-state activity in the presence of Ca2+. Furthermore, Ca2+ is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg2+. We therefore hypothesize that the physiological metal ions Mg2+ and Ca2+ both play a role in kinase activity and regulation. Since PKA is localized close to calcium channels and may render PKA activity susceptible to Ca2+, our data provide a possible mechanism for novel crosstalk between cAMP and calcium signaling. © 2015 American Chemical Society.

Munari F.,Max Planck Institute for Biophysical Chemistry | Soeroes S.,Max Planck Institute for Biophysical Chemistry | Soeroes S.,Oxford Nanopore Technologies | Zenn H.M.,Biaffin GmbH and Co. KG | And 16 more authors.
Journal of Biological Chemistry | Year: 2012

Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1β to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1β with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1β-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Hermann J.S.,University of Kassel | Skroblin P.,Anchored Signaling | Bertinetti D.,University of Kassel | Hanold L.E.,University of Georgia | And 8 more authors.
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2015

Protein kinase activity is regulated not only by direct strategies affecting activity but also by spatial and temporal regulatory mechanisms. Kinase signaling pathways are coordinated by scaffolding proteins that orchestrate the assembly of multi-protein complexes. One family of such scaffolding proteins are the A-kinase anchoring proteins (AKAPs). AKAPs share the commonality of binding cAMP-dependent protein kinase (PKA). In addition, they bind further signaling proteins and kinase substrates and tether such multi-protein complexes to subcellular locations. The A-kinase binding (AKB) domain of AKAPs typically contains a conserved helical motif that interacts directly with the dimerization/docking (D/D) domain of the regulatory subunits of PKA. Based on a pull-down proteomics approach, we identified neurochondrin (neurite-outgrowth promoting protein) as a previously unidentified AKAP. Here, we show that neurochondrin interacts directly with PKA through a novel mechanism that involves two distinct binding regions. In addition, we demonstrate that neurochondrin has strong isoform selectivity towards the RIIα subunit of PKA with nanomolar affinity. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases © 2015 Elsevier B.V. All rights reserved.

Oesterreich B.,University of Würzburg | Lorenz B.,University of Würzburg | Schmitter T.,University of Würzburg | Kontermann R.,University of Stuttgart | And 5 more authors.
Human Vaccines and Immunotherapeutics | Year: 2014

Multi-antigen immunotherapy approaches against Staphylococcus aureus are expected to have the best chance of clinical success when used in combinatorial therapy, potentially incorporating opsonic killing of bacteria and toxin neutralization. We recently reported the development of a murine monoclonal antibody specific for the immunodominant staphylococcal antigen A (IsaA), which showed highly efficient staphylococcal killing in experimental infection models of S. aureus. If IsaA-specific antibodies are to be used as a component of combination therapy in humans, the binding specificity and biological activity of the humanized variant must be preserved. Here, we describe the functional characterization of a humanized monoclonal IgG1 variant designated, hUK-66. The humanized antibody showed comparable binding kinetics to those of its murine parent, and recognized the target antigen IsaA on the surface of clinically relevant S. aureus lineages. Furthermore, hUK-66 enhances the killing of S. aureus in whole blood (a physiological environment) samples from healthy subjects and patients prone to staphylococcal infections such as diabetes and dialysis patients, and patients with generalized artery occlusive disease indicating no interference with already present natural antibodies. Taken together, these data indicate that hUK-66 mediates bacterial killing even in high risk patients and thus, could play a role for immunotherapy strategies to combat severe S. aureus infections. © 2014 Landes Bioscience.

Fender A.,Uppsala University | Elf J.,Uppsala University | Elf J.,Science for Life Laboratory | Hampel K.,Biaffin GmbH and Co. KG | And 3 more authors.
Genes and Development | Year: 2010

Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar Kd values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1- 2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentration-driven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA-Hfq complexes are ≈1 min. We propose that competitor RNA associates transiently with RNA-Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the "strong binding-high turn-over" paradox and permits efficient use of the Hfq pool. © 2010 by Cold Spring Harbor Laboratory Press.

Schmidt J.,Helmholtz Center for Infection Research | Musken M.,Helmholtz Center for Infection Research | Becker T.,Helmholtz Center for Infection Research | Magnowska Z.,Helmholtz Center for Infection Research | And 6 more authors.
PLoS ONE | Year: 2011

The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa. © 2011 Schmidt et al.

Grancha S.,Grifols | Ortiz A.M.,Grifols | Maranon C.,Grifols | Hampel K.,Biaffin GmbH and Co KG | And 4 more authors.
Haemophilia | Year: 2012

The presence of VWF in plasma-derived FVIII (pdFVIII/VWF) products has been pointed out as a key difference with recombinant FVIII (rFVIII) products with regard to immunogenicity. A Surface Plasmon Resonance (SPR) study was designed to characterize in detail the interaction between anti-FVIII (IgGs) from a severe haemophilia A patient, and FVIII from concentrates of different sources. Full-length rFVIII (preincubated or not with purified VWF), B domain-deleted (BDD)-rFVIII and pdFVIII/VWF were analysed. To ensure reproducible conditions for accurate determination of kinetic constants, a capture-based assay format was developed using protein G surfaces for specific and reversible coupling of endogenous anti-FVIII antibodies. Concentration ranges (nm) of FVIII products tested were 9-0.03 (rFVIII) and 6-0.024 (pdFVIII/VWF). The association with antibodies was monitored for 3-5 min, whereas dissociation of the complex was followed for 5-20-240 min. A strong interaction of rFVIII and BDD-rFVIII with patient's IgG was detected with the K D values in the low picomolar range (5.9 ± 3.0 and 12.7 ± 6.9 pm, respectively) and very slow dissociation rates, while pdFVIII/VWF showed only marginal binding signals. The VWF complexed rFVIII displayed reduced binding signals compared with uncomplexed rFVIII, but the K D was still in the picomolar range (4.1 ± 1.9 pm) indicating insufficient complex formation. rFVIII, alone or bound to exogenously added VWF, showed high affinity for anti-FVIII IgGs from a severe haemophilia A patient whereas pdFVIII/VWF did not. These results are in agreement with those studies that point towards rFVIII concentrates to be more immunogenic than pdFVIII concentrates. © 2012 Blackwell Publishing Ltd.

Duvel J.,Helmholtz Center for Infection Research | Bertinetti D.,University of Kassel | Moller S.,University of Kassel | Schwede F.,Biolog Life Science Institute | And 9 more authors.
Journal of Microbiological Methods | Year: 2012

In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen . Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2'-aminohexylcarbamoyl-c-di-GMP (2'-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2'-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules . in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network. © 2011 Elsevier B.V..

Kummer L.,University of Zürich | Hsu C.-W.,University of North Carolina at Chapel Hill | Dagliyan O.,University of North Carolina at Chapel Hill | MacNevin C.,University of North Carolina at Chapel Hill | And 5 more authors.
Chemistry and Biology | Year: 2013

Investigation of protein activation in living cells is fundamental to understanding how proteins are influenced by the full complement of upstream regulators they experience. Here, we describe the generation of a biosensor based on the DARPin binding scaffold suited for intracellular applications. Combining library selection and knowledge-based design, we created an ERK activity biosensor by derivatizing a DARPin specific for phosphorylated ERK with a solvatochromatic merocyanine dye, whose fluorescence increases upon pERK binding. The biosensor specifically responded to pERK2, recognized by its conformation, but not to ERK2 or other closely related mitogen-activated kinases tested. Activated endogenous ERK was visualized in mouse embryo fibroblasts, revealing greater activation in the nucleus, perinuclear regions, and especially the nucleoli. The DARPin-based biosensor will serve as a useful tool for studying biological functions of ERK in vitro and in vivo. © 2013 Elsevier Ltd. All rights reserved.

Lorenz U.,University of Würzburg | Lorenz B.,University of Würzburg | Schmitter T.,University of Würzburg | Streker K.,University of Würzburg | And 5 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2011

Staphylococcus aureus is the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase of S. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized all S. aureus strains tested, including hospital-acquired and community-acquired methicillin-resistant S. aureus strains. Therapeutic efficacy in vivo in mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection. Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases of S. aureus infections. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

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