Bastian Zimmermann - Academia.edu (original) (raw)

Papers by Bastian Zimmermann

Research paper thumbnail of Structural insights into heme binding to IL-36α proinflammatory cytokine

Scientific Reports

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recent... more Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulat...

Research paper thumbnail of Calcium and zinc tune autoinflammatory Toll-like receptor 4 signaling by S100A12

The Journal of allergy and clinical immunology, Jan 3, 2018

This study was supported by grants from the intramural innovative medical research program of Mue... more This study was supported by grants from the intramural innovative medical research program of Muenster University medical faculty (grant no. KE121201 to C.K.) and the German Research Foundation (DFG, grant no. WI3176/2-1 to H.W. and grant no. Fo354/3-1 to D.F.). Disclosure of potential conflict of interest: C. Kessel & D. Foell have filed a patent application on ''means and methods for diagnosing and treating inflammatory disorders'' regarding proinflammatory S100A12 homomultimers (WO 2016/178154 A1). The rest of the authors declare that they have no relevant conflicts of interests.

Research paper thumbnail of Divalent metal ions control activity and inhibition of protein kinases

Metallomics : integrated biometal science, Jan 15, 2017

Protein kinases are key enzymes in the regulation of eukaryotic signal transduction. As metalloen... more Protein kinases are key enzymes in the regulation of eukaryotic signal transduction. As metalloenzymes they employ divalent cations for catalysis and regulation. We used the catalytic (C) subunit of cAMP-dependent protein kinase (PKA) as a model protein to investigate the role of a variety of physiologically or pathophysiologically relevant divalent metal ions in distinct steps within the catalytic cycle. It is established that divalent metal ions play a crucial role in co-binding of nucleotides and also assist in catalysis. Our studies reveal that besides the physiologically highly relevant magnesium, metals like zinc and manganese can assist in phosphoryl transfer, however, only a few support efficient substrate turnover (turnover catalysis). Those trace metals allow for substrate binding and phosphotransfer but hamper product release. We further established the unique role of magnesium as the common biologically relevant divalent metal ideally enabling (co-) substrate binding and...

Research paper thumbnail of Untersuchung von Metallionen induzierten Konformationsänderungen in Proteinkinasen mittels Surface Plasmon Resonance

Research paper thumbnail of Fusion Proteins, Method for the Production and Use Thereof

Research paper thumbnail of Current developments for the in vitro characterization of the PKA system

Research paper thumbnail of Quantification of cAMP antagonist action in vitro and in living cells

European Journal of Cell Biology, Jul 5, 2006

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonist... more cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET(2), performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ialpha PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.

Research paper thumbnail of High-affinity AKAP7δ-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

Biochemical Journal, 2006

Research paper thumbnail of Dynamic and flexible H3K9me3 bridging via HP1β dimerization establishes a plastic state of condensed chromatin

Nature communications, Jan 19, 2016

Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP... more Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1β is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1β bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1β genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 prot...

Research paper thumbnail of Fluoreszenz-und Biolumineszenzmethoden in der Proteinanalytik

Research paper thumbnail of Applications of biomolecular interaction analysis in drug development

TARGETS, 2002

Biomolecular interaction analysis (BIA) is now utilised increasingly in drug development to kinet... more Biomolecular interaction analysis (BIA) is now utilised increasingly in drug development to kinetically characterise binding events of relevant components. BIA thereby covers a broad range of applications in early ADME (adsorption, distribution, metabolism and excretion), secondary screens, functional and metabolic assays and in assay development.This versatile technology allows measurements in real time without the need of labelling. A wide range of affinities can be characterised in a nearnative state, with high reproducibility, high sensitivity and low sample consumption. However, limitations due to experimental design, and limitations in sample throughput, may occur. Future developments of BIA aim at parallelisation and automatisation and at coupling to platform technologies already established in the drug development process.

Research paper thumbnail of PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent pr... more The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 mol/(min⅐mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RI ␣) of cAMP-dependent protein kinase (K D ‫؍‬ 10 nM) and the heat-stable protein kinase inhibitor (K D ‫؍‬ 15 nM), but not with the type II regulatory subunit (RII ␣ , K D ‫؍‬ 2.3 M) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RI ␣ subunit and by protein kinase inhibitor in vitro, but only weakly by the RII ␣ subunit. The inhibition by the RI ␣ subunit is reversed by addition of nanomolar concentrations of cAMP (K a ‫؍‬ 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the C ␣ subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RII ␣ subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RI ␣ subunit and by phosphorylating the RII ␣ subunit in the absence of cAMP.

Research paper thumbnail of Fluoreszenz-und Biolumineszenzmethoden in der Proteinanalytik

Research paper thumbnail of Current Developments for the In Vitro Characterization of Protein Interactions

Methods and Principles in Medicinal Chemistry, 2006

... Daniela Moll, Bastian Zimmermann, Frank Gesellchen and Friedrich W. Herberg ... S, indicating... more ... Daniela Moll, Bastian Zimmermann, Frank Gesellchen and Friedrich W. Herberg ... S, indicating that binding is driven by hydrophobic interactions, but with an unfavorable ∆H, may be poorly soluble and conformational restraints may lead to a lack of adaptability (Luque and Freire ...

Research paper thumbnail of Direct Optical Detection of Protein–Ligand Interactions

Protein-Ligand Interactions, 2005

Direct optical detection provides an excellent means to investigate interactions of molecules in ... more Direct optical detection provides an excellent means to investigate interactions of molecules in biological systems. The dynamic equilibria inherent to these systems can be described in greater detail by recording the kinetics of a biomolecular interaction. Optical biosensors allow direct detection of interaction patterns without the need for labeling. An overview covering several commercially available biosensors is given, with a focus on instruments based on surface plasmon resonance (SPR) and reflectometric interference spectroscopy (RIFS). Potential assay formats and experimental design, appropriate controls, and calibration procedures, especially when handling low molecular weight substances, are discussed. The single steps of an interaction analysis combined with practical tips for evaluation, data processing, and interpretation of kinetic data are described in detail. In a practical example, a step-by-step procedure for the analysis of a low molecular weight compound interaction with serum protein, determined on a commercial SPR sensor, is presented.

Research paper thumbnail of Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1  with the Nucleosome

Journal of Biological Chemistry, 2012

Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) ma... more 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.

Research paper thumbnail of Biomedical application of SPR biosensors Biomedical application of surface plasmon resonance biosensors (review)

Research paper thumbnail of Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface

Protein Science, 2008

All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of ... more All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of CAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (AI-14) as well as the entire N-terminal segment (AI-39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(Al-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decreases in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(A1-39) as a fusion protein with glutathione-S-transferase (GST). GST-rC(Al-39), although its thermostability (T,) was decreased by 12.5 "C, was catalytically similar to wild-type C and was inhibited by both the type I and I1 R-subunits and the heat-stable protein kinase inhibitor (PKI). The T, for holoenzyme I1 formed with GST-rC(A1-39) was 16.5 "C greater than the T, for free GST-rC(A1-39), and the &(CAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large lobe so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.

Research paper thumbnail of Quantification of cAMP antagonist action in vitro and in living cells

European Journal of Cell Biology, 2006

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonist... more cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET 2 , performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ia PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.

Research paper thumbnail of Plasma Protein Binding Properties to Immobilized Heparin and Heparin?Albumin Conjugate

Artificial Organs, 2007

Selective adhesion of plasma proteins to immobilized heparin is considered to be beneficial regar... more Selective adhesion of plasma proteins to immobilized heparin is considered to be beneficial regarding hemocompatibility of foreign materials in contact with blood. Prothrombin, thrombin, antithrombin III (AT3), and fibrinogen were selected for analysis in an experimental model. Biomolecular interaction analysis employing surface plasmon resonance was utilized to record and analyze their binding properties in real time. Biotinylated heparin, heparin-albumin conjugate, and albumin, respectively, were immobilized onto streptavidin-coated sensors as ligands. Prothrombin did not bind to any of the ligand surfaces and no specific binding of any of the plasma proteins to albumin was observed. Binding kinetics of thrombin to heparin and to heparin-albumin conjugate were calculated using two different methods. For heparin, identical KD (equilibrium dissociation constant) values of 61 ¥ 10 -9 M were obtained with both methods. For the conjugate, only slightly different KD values of 111 ¥ 10 -9 and 104 ¥ 10 -9 M, respectively, were calculated. The affinity of thrombin toward the heparin-coated surface proved to be higher than its affinity toward the heparin conjugate. The binding pattern of AT3 to both heparin and heparinalbumin conjugate, although specific, was biphasic, possibly due to a conformational change during the binding process. Steady-state kinetic analysis revealed a KD value of 281 Ϯ 24 ¥ 10 -9 M for the heparin surface. For the conjugate surface, a KD of 53 Ϯ 5 ¥ 10 -9 M was calculated, indicating a higher affinity toward heparin-albumin conjugate. A high-affinity binding of fibrinogen to high-density surfaces of both heparin and the conjugate was observed. However, as binding to low-density surfaces was considerably reduced, specificity remained uncertain.

Research paper thumbnail of Structural insights into heme binding to IL-36α proinflammatory cytokine

Scientific Reports

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recent... more Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulat...

Research paper thumbnail of Calcium and zinc tune autoinflammatory Toll-like receptor 4 signaling by S100A12

The Journal of allergy and clinical immunology, Jan 3, 2018

This study was supported by grants from the intramural innovative medical research program of Mue... more This study was supported by grants from the intramural innovative medical research program of Muenster University medical faculty (grant no. KE121201 to C.K.) and the German Research Foundation (DFG, grant no. WI3176/2-1 to H.W. and grant no. Fo354/3-1 to D.F.). Disclosure of potential conflict of interest: C. Kessel & D. Foell have filed a patent application on ''means and methods for diagnosing and treating inflammatory disorders'' regarding proinflammatory S100A12 homomultimers (WO 2016/178154 A1). The rest of the authors declare that they have no relevant conflicts of interests.

Research paper thumbnail of Divalent metal ions control activity and inhibition of protein kinases

Metallomics : integrated biometal science, Jan 15, 2017

Protein kinases are key enzymes in the regulation of eukaryotic signal transduction. As metalloen... more Protein kinases are key enzymes in the regulation of eukaryotic signal transduction. As metalloenzymes they employ divalent cations for catalysis and regulation. We used the catalytic (C) subunit of cAMP-dependent protein kinase (PKA) as a model protein to investigate the role of a variety of physiologically or pathophysiologically relevant divalent metal ions in distinct steps within the catalytic cycle. It is established that divalent metal ions play a crucial role in co-binding of nucleotides and also assist in catalysis. Our studies reveal that besides the physiologically highly relevant magnesium, metals like zinc and manganese can assist in phosphoryl transfer, however, only a few support efficient substrate turnover (turnover catalysis). Those trace metals allow for substrate binding and phosphotransfer but hamper product release. We further established the unique role of magnesium as the common biologically relevant divalent metal ideally enabling (co-) substrate binding and...

Research paper thumbnail of Untersuchung von Metallionen induzierten Konformationsänderungen in Proteinkinasen mittels Surface Plasmon Resonance

Research paper thumbnail of Fusion Proteins, Method for the Production and Use Thereof

Research paper thumbnail of Current developments for the in vitro characterization of the PKA system

Research paper thumbnail of Quantification of cAMP antagonist action in vitro and in living cells

European Journal of Cell Biology, Jul 5, 2006

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonist... more cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET(2), performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ialpha PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.

Research paper thumbnail of High-affinity AKAP7δ-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

Biochemical Journal, 2006

Research paper thumbnail of Dynamic and flexible H3K9me3 bridging via HP1β dimerization establishes a plastic state of condensed chromatin

Nature communications, Jan 19, 2016

Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP... more Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1β is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1β bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1β genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 prot...

Research paper thumbnail of Fluoreszenz-und Biolumineszenzmethoden in der Proteinanalytik

Research paper thumbnail of Applications of biomolecular interaction analysis in drug development

TARGETS, 2002

Biomolecular interaction analysis (BIA) is now utilised increasingly in drug development to kinet... more Biomolecular interaction analysis (BIA) is now utilised increasingly in drug development to kinetically characterise binding events of relevant components. BIA thereby covers a broad range of applications in early ADME (adsorption, distribution, metabolism and excretion), secondary screens, functional and metabolic assays and in assay development.This versatile technology allows measurements in real time without the need of labelling. A wide range of affinities can be characterised in a nearnative state, with high reproducibility, high sensitivity and low sample consumption. However, limitations due to experimental design, and limitations in sample throughput, may occur. Future developments of BIA aim at parallelisation and automatisation and at coupling to platform technologies already established in the drug development process.

Research paper thumbnail of PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent pr... more The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 mol/(min⅐mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RI ␣) of cAMP-dependent protein kinase (K D ‫؍‬ 10 nM) and the heat-stable protein kinase inhibitor (K D ‫؍‬ 15 nM), but not with the type II regulatory subunit (RII ␣ , K D ‫؍‬ 2.3 M) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RI ␣ subunit and by protein kinase inhibitor in vitro, but only weakly by the RII ␣ subunit. The inhibition by the RI ␣ subunit is reversed by addition of nanomolar concentrations of cAMP (K a ‫؍‬ 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the C ␣ subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RII ␣ subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RI ␣ subunit and by phosphorylating the RII ␣ subunit in the absence of cAMP.

Research paper thumbnail of Fluoreszenz-und Biolumineszenzmethoden in der Proteinanalytik

Research paper thumbnail of Current Developments for the In Vitro Characterization of Protein Interactions

Methods and Principles in Medicinal Chemistry, 2006

... Daniela Moll, Bastian Zimmermann, Frank Gesellchen and Friedrich W. Herberg ... S, indicating... more ... Daniela Moll, Bastian Zimmermann, Frank Gesellchen and Friedrich W. Herberg ... S, indicating that binding is driven by hydrophobic interactions, but with an unfavorable ∆H, may be poorly soluble and conformational restraints may lead to a lack of adaptability (Luque and Freire ...

Research paper thumbnail of Direct Optical Detection of Protein–Ligand Interactions

Protein-Ligand Interactions, 2005

Direct optical detection provides an excellent means to investigate interactions of molecules in ... more Direct optical detection provides an excellent means to investigate interactions of molecules in biological systems. The dynamic equilibria inherent to these systems can be described in greater detail by recording the kinetics of a biomolecular interaction. Optical biosensors allow direct detection of interaction patterns without the need for labeling. An overview covering several commercially available biosensors is given, with a focus on instruments based on surface plasmon resonance (SPR) and reflectometric interference spectroscopy (RIFS). Potential assay formats and experimental design, appropriate controls, and calibration procedures, especially when handling low molecular weight substances, are discussed. The single steps of an interaction analysis combined with practical tips for evaluation, data processing, and interpretation of kinetic data are described in detail. In a practical example, a step-by-step procedure for the analysis of a low molecular weight compound interaction with serum protein, determined on a commercial SPR sensor, is presented.

Research paper thumbnail of Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1  with the Nucleosome

Journal of Biological Chemistry, 2012

Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) ma... more 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.

Research paper thumbnail of Biomedical application of SPR biosensors Biomedical application of surface plasmon resonance biosensors (review)

Research paper thumbnail of Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface

Protein Science, 2008

All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of ... more All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of CAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (AI-14) as well as the entire N-terminal segment (AI-39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(Al-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decreases in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(A1-39) as a fusion protein with glutathione-S-transferase (GST). GST-rC(Al-39), although its thermostability (T,) was decreased by 12.5 "C, was catalytically similar to wild-type C and was inhibited by both the type I and I1 R-subunits and the heat-stable protein kinase inhibitor (PKI). The T, for holoenzyme I1 formed with GST-rC(A1-39) was 16.5 "C greater than the T, for free GST-rC(A1-39), and the &(CAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large lobe so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.

Research paper thumbnail of Quantification of cAMP antagonist action in vitro and in living cells

European Journal of Cell Biology, 2006

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonist... more cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET 2 , performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ia PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.

Research paper thumbnail of Plasma Protein Binding Properties to Immobilized Heparin and Heparin?Albumin Conjugate

Artificial Organs, 2007

Selective adhesion of plasma proteins to immobilized heparin is considered to be beneficial regar... more Selective adhesion of plasma proteins to immobilized heparin is considered to be beneficial regarding hemocompatibility of foreign materials in contact with blood. Prothrombin, thrombin, antithrombin III (AT3), and fibrinogen were selected for analysis in an experimental model. Biomolecular interaction analysis employing surface plasmon resonance was utilized to record and analyze their binding properties in real time. Biotinylated heparin, heparin-albumin conjugate, and albumin, respectively, were immobilized onto streptavidin-coated sensors as ligands. Prothrombin did not bind to any of the ligand surfaces and no specific binding of any of the plasma proteins to albumin was observed. Binding kinetics of thrombin to heparin and to heparin-albumin conjugate were calculated using two different methods. For heparin, identical KD (equilibrium dissociation constant) values of 61 ¥ 10 -9 M were obtained with both methods. For the conjugate, only slightly different KD values of 111 ¥ 10 -9 and 104 ¥ 10 -9 M, respectively, were calculated. The affinity of thrombin toward the heparin-coated surface proved to be higher than its affinity toward the heparin conjugate. The binding pattern of AT3 to both heparin and heparinalbumin conjugate, although specific, was biphasic, possibly due to a conformational change during the binding process. Steady-state kinetic analysis revealed a KD value of 281 Ϯ 24 ¥ 10 -9 M for the heparin surface. For the conjugate surface, a KD of 53 Ϯ 5 ¥ 10 -9 M was calculated, indicating a higher affinity toward heparin-albumin conjugate. A high-affinity binding of fibrinogen to high-density surfaces of both heparin and the conjugate was observed. However, as binding to low-density surfaces was considerably reduced, specificity remained uncertain.