75 results on '"Kötting C"'
Search Results
2. Virtual staining of colon cancer tissue by label-free Raman micro-spectroscopy
- Author
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Petersen, D., primary, Mavarani, L., additional, Niedieker, D., additional, Freier, E., additional, Tannapfel, A., additional, Kötting, C., additional, Gerwert, K., additional, and El-Mashtoly, S. F., additional
- Published
- 2017
- Full Text
- View/download PDF
3. Local Mode Analysis: Decoding IR Spectra by Visualizing Molecular Details.
- Author
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Massarczyk, M., Rudack, T., Schlitter, J., Kuhne, J., Kötting, C., and Gerwert, K.
- Published
- 2017
- Full Text
- View/download PDF
4. Kosten-Nutzen-Bewertung als Basis einer zentralen Preisregulierung für verschreibungspflichtige Arzneimittel
- Author
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May, U., primary, Kötting, C., additional, Klaucke, L., additional, Greß, S., additional, and Wasem, J., additional
- Published
- 2009
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5. Rabattverträge in der gesetzlichen Krankenversicherung – Auswirkungen einer Oligopolisierung des generikafähigen Arzneimittelmarkts
- Author
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Greß, S., primary, Kötting, C., additional, May, U., additional, and Wasem, J., additional
- Published
- 2009
- Full Text
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6. Decentralized prize negotiations in German social health insurance -- consequences of oligopolies on the market for generic prescription drugs.
- Author
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Greß S, Kötting C, May U, and Wasem J
- Published
- 2009
7. A Clickable Photosystem I, Ferredoxin, and Ferredoxin NADP + Reductase Fusion System for Light-Driven NADPH Regeneration.
- Author
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Medipally H, Mann M, Kötting C, van Berkel WJH, and Nowaczyk MM
- Subjects
- NADP metabolism, Ferredoxin-NADP Reductase metabolism, Ferredoxins metabolism, Electron Transport, Photosystem I Protein Complex chemistry, Photosystem I Protein Complex metabolism, Synechocystis
- Abstract
Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b
6 f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI-Fd and PSI-Fd-FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI-PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7-PSI was coupled with the E7-Fd or E7-Fd-FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)- Published
- 2023
- Full Text
- View/download PDF
8. ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy.
- Author
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Mann D, Labudda K, Zimmermann S, Vocke KU, Gasper R, Kötting C, and Hofmann E
- Subjects
- Spectroscopy, Fourier Transform Infrared, Hydrolysis, Adenosine Triphosphate metabolism, Escherichia coli metabolism, Bacterial Proteins metabolism, Escherichia coli Proteins metabolism
- Abstract
The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k
1 = 0.49 ± 0.28 s-1 and k2 = 0.014 ± 0.003 s-1 , respectively. We further verified these rates with photocaged NPE cg AppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis ( k2 < 2 × 10-4 s-1 ). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)- Published
- 2023
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9. Time-resolved spectroscopic and electrophysiological data reveal insights in the gating mechanism of anion channelrhodopsin.
- Author
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Dreier MA, Althoff P, Norahan MJ, Tennigkeit SA, El-Mashtoly SF, Lübben M, Kötting C, Rudack T, and Gerwert K
- Subjects
- Light, Optogenetics, Spectrophotometry, Anions chemistry, Channelrhodopsins physiology, Cryptophyta physiology, Electrophysiological Phenomena, Ion Channel Gating
- Abstract
Channelrhodopsins are widely used in optogenetic applications. High photocurrents and low current inactivation levels are desirable. Two parallel photocycles evoked by different retinal conformations cause cation-conducting channelrhodopsin-2 (CrChR2) inactivation: one with efficient conductivity; one with low conductivity. Given the longer half-life of the low conducting photocycle intermediates, which accumulate under continuous illumination, resulting in a largely reduced photocurrent. Here, we demonstrate that for channelrhodopsin-1 of the cryptophyte Guillardia theta (GtACR1), the highly conducting C = N-anti-photocycle was the sole operating cycle using time-resolved step-scan FTIR spectroscopy. The correlation between our spectroscopic measurements and previously reported electrophysiological data provides insights into molecular gating mechanisms and their role in the characteristic high photocurrents. The mechanistic importance of the central constriction site amino acid Glu-68 is also shown. We propose that canceling out the poorly conducting photocycle avoids the inactivation observed in CrChR2, and anticipate that this discovery will advance the development of optimized optogenetic tools.
- Published
- 2021
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10. Microsecond-Resolved Infrared Spectroscopy on Nonrepetitive Protein Reactions by Applying Caged Compounds and Quantum Cascade Laser Frequency Combs.
- Author
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Norahan MJ, Horvath R, Woitzik N, Jouy P, Eigenmann F, Gerwert K, and Kötting C
- Subjects
- Spectrophotometry, Infrared, Spectroscopy, Fourier Transform Infrared, Lasers, Semiconductor
- Abstract
Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about 10 ms with 16 cm
-1 resolution), step-scan FTIR spectroscopy reaches a time resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of nonrepetitive reactions, e.g., induced by caged compounds, were limited to the millisecond time domain. The advent of dual-comb quantum cascade laser now allows for a rapid reaction monitoring in the microsecond time domain. Here, we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound-induced reactions monitored by FTIR and dual-comb spectroscopy by applying the new technique to the α subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gαi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with a 4 μs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable microsecond time regime for nonrepetitive biological systems including all GTPases and ATPases.- Published
- 2021
- Full Text
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11. The Ras dimer structure.
- Author
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Rudack T, Teuber C, Scherlo M, Güldenhaupt J, Schartner J, Lübben M, Klare J, Gerwert K, and Kötting C
- Abstract
Oncogenic mutated Ras is a key player in cancer, but despite intense and expensive approaches its catalytic center seems undruggable. The Ras dimer interface is a possible alternative drug target. Dimerization at the membrane affects cell growth signal transduction. In vivo studies indicate that preventing dimerization of oncogenic mutated Ras inhibits uncontrolled cell growth. Conventional computational drug-screening approaches require a precise atomic dimer model as input to successfully access drug candidates. However, the proposed dimer structural models are controversial. Here, we provide a clear-cut experimentally validated N-Ras dimer structural model. We incorporated unnatural amino acids into Ras to enable the binding of labels at multiple positions via click chemistry. This labeling allowed the determination of multiple distances of the membrane-bound Ras-dimer measured by fluorescence and electron paramagnetic resonance spectroscopy. In combination with protein-protein docking and biomolecular simulations, we identified key residues for dimerization. Site-directed mutations of these residues prevent dimer formation in our experiments, proving our dimer model to be correct. The presented dimer structure enables computational drug-screening studies exploiting the Ras dimer interface as an alternative drug target., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2021
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12. Immune-Stimulatory Effects of Curcumin on the Tumor Microenvironment in Head and Neck Squamous Cell Carcinoma.
- Author
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Kötting C, Hofmann L, Lotfi R, Engelhardt D, Laban S, Schuler PJ, Hoffmann TK, Brunner C, and Theodoraki MN
- Abstract
Curcumin is known to have immune-modulatory and antitumor effects by interacting with more than 30 different proteins. An important feature of curcumin is the inhibition of nuclear factor kappa of activated B-cells (NF-κB). Here, we evaluate the potential of curcumin to reverse the epithelial to mesenchymal transition (EMT) of head and neck squamous cell carcinoma (HNSCC) cells as a part of tumor escape mechanisms. We examined the impact of curcumin on the expression of different pro- and antitumoral chemokines in ex vivo HNSCC tumor tissue and primary macrophage cultures. Further, we evaluated the combinatorial effect of curcumin and toll-like receptor 3 (TLR3) agonist Poly I:C (PIC) on NF-κB inhibition and regulatory T-cell (Treg) attraction. Mesenchymal markers were significantly reduced in cancer specimens after incubation with curcumin, with simultaneous reduction of key transcription factors of EMT, Snail, and Twist. Furthermore, a decrease of the Treg-attracting chemokine CCL22 was observed. Additionally, curcumin-related inhibition of NF-κB nuclear translocation was evident. The combination of PIC with curcumin resulted in further NF-κB inhibition, whereas PIC alone contrarily resulted in NF-κB activation. Furthermore, curcumin was more effective in inhibiting PIC-dependent NF-κB activation and Treg attraction compared to known NF-κB inhibitors BAY 11-7082 or caffeic acid phenethyl ester (CAPE). The presented results show, for the first time, the immune-modulating effects of curcumin in HNSCC, with potent inhibition of the Treg-attracting effects of PIC. Hence, curcumin presents a promising drug in cancer therapy as a supplement to already established treatments.
- Published
- 2021
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13. Reversible Immuno-Infrared Sensor for the Detection of Alzheimer's Disease Related Biomarkers.
- Author
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Budde B, Schartner J, Tönges L, Kötting C, Nabers A, and Gerwert K
- Subjects
- Alzheimer Disease cerebrospinal fluid, Amyloid beta-Peptides immunology, Antibodies, Immobilized immunology, Antibodies, Monoclonal immunology, Bacterial Proteins chemistry, Biomarkers cerebrospinal fluid, Germanium chemistry, Humans, Immunoglobulin G immunology, Peptide Fragments immunology, Reproducibility of Results, Spectroscopy, Fourier Transform Infrared instrumentation, Spectroscopy, Fourier Transform Infrared methods, Staphylococcal Protein A chemistry, tau Proteins immunology, Alzheimer Disease diagnosis, Amyloid beta-Peptides cerebrospinal fluid, Peptide Fragments cerebrospinal fluid, tau Proteins cerebrospinal fluid
- Abstract
The development of biosensors for medical purposes is a growing field. An immuno-infrared biosensor for the preclinical detection of Alzheimer's disease (AD) in body fluids was developed. The key element of this sensor is an ATR crystal with chemically modified surface to catch the biomarker out of the body fluid. So far, the immuno-infrared sensor can be used only once and requires time-consuming steps of sensor exchange, sensor cleaning, and novel surface functionalization. Here, we developed an immuno-infrared sensor providing a reusable surface and showcase its performance by the detection of the AD biomarker proteins Aβ and Tau in human cerebrospinal fluid (CSF). The sensor surface is covalently coated with the immunoglobulin binding proteins Protein A or Protein G. These were employed for noncovalent immobilization of antibodies and the subsequent immobilization and analysis of their antigens. The reversible antibody immobilization can be repeated several times with the same or different antibodies. Further, the more specific binding of the antibody via its Fc region instead of the conventional NHS coupling leads to a 3-4-fold higher antigen binding capacity of the antibody. Thus, the throughput, sensitivity, and automation capacity of the immuno-infrared biosensor are significantly increased as compared to former immuno-infrared assays. This immuno-sensor can be used with any antibody that binds to Protein A or Protein G.
- Published
- 2019
- Full Text
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14. GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases.
- Author
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Calixto AR, Moreira C, Pabis A, Kötting C, Gerwert K, Rudack T, and Kamerlin SCL
- Subjects
- Animals, Catalytic Domain, Enzyme Activation, GTP Phosphohydrolases chemistry, Humans, Hydrolysis, Models, Molecular, GTP Phosphohydrolases metabolism, Guanosine Triphosphate metabolism
- Abstract
GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the G
αi subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2 PO4 - and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) "undruggable" GTPases like Ras.- Published
- 2019
- Full Text
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15. Monitoring transient events in infrared spectra using local mode analysis.
- Author
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Massarczyk M, Schlitter J, Kötting C, Rudack T, and Gerwert K
- Subjects
- Deuterium chemistry, Molecular Dynamics Simulation, Protons, Quantum Theory, Software, Malondialdehyde chemistry, Spectroscopy, Fourier Transform Infrared methods
- Abstract
Time-resolved Fourier transformed infrared (FTIR) spectroscopy of chemical reactions is highly sensitive to minimal spatiotemporal changes. Structural features are decoded and represented in a comprehensible manner by combining FTIR spectroscopy with biomolecular simulations. Local mode analysis (LMA) is a tool to connect molecular motion based on a quantum mechanics simulation with infrared (IR) spectral features and vice versa. Here, we present the python-based software tool of LMA and demonstrate the novel feature of LMA to extract transient structural details and identify the related IR spectra at the case example of malonaldehyde (MA). Deuterated MA exists in two almost equally populated tautomeric states separated by a low barrier for proton transfer so IR spectra represent a mixture of both states. By state-dependent LMA, we obtain pure spectra for each tautomeric state occurring within the quantum mechanics trajectory. By time-resolved LMA, we obtain a clear view of the transition between states in the spectrum. Through local mode decomposition and the band-pass filter, marker bands for each state are identified. Thus, LMA is beneficial to analyze the experimental spectra based on a mixture of states by determining the individual contributions to the spectrum and motion of each state., (© 2018 Wiley Periodicals, Inc.)
- Published
- 2018
- Full Text
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16. Label-free identification of myopathological features with coherent anti-Stokes Raman scattering.
- Author
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Niedieker D, GrosserÜschkamp F, Schreiner A, Barkovits K, Kötting C, Marcus K, Gerwert K, and Vorgerd M
- Subjects
- Child, Preschool, Female, Humans, Infant, Male, Middle Aged, Spectrum Analysis, Raman methods, Glycogen Storage Disease Type V diagnostic imaging, Glycogen Storage Disease Type V metabolism, Nonlinear Optical Microscopy methods
- Abstract
Introduction: The aim of this study was the label-free identification of distinct myopathological features with coherent anti-Stokes Raman scattering (CARS) imaging, which leaves the sample intact for further analysis., Methods: The protein distribution was determined without labels by CARS at 2,930 cm
-1 and was compared with the results of standard histological staining., Results: CARS imaging allowed the visualization of glycogen accumulation in glycogen storage disease type 5 (McArdle disease) and of internal nuclei in centronuclear myopathy. CARS identified an inhomogeneous protein distribution within muscle fibers in sporadic inclusion body myositis that was not shown with standard staining. In Duchenne muscular dystrophy, evidence for a higher protein content at the border of hypercontracted fibers was detected., Discussion: CARS enables the label-free identification of distinct myopathological features, possibly paving the way for subsequent proteomic, metabolic, and genomic analyses. Muscle Nerve 58: 457-460, 2018., (© 2018 Wiley Periodicals, Inc.)- Published
- 2018
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17. Ligand-Induced Conformational Changes in HSP90 Monitored Time Resolved and Label Free-Towards a Conformational Activity Screening for Drug Discovery.
- Author
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Güldenhaupt J, Amaral M, Kötting C, Schartner J, Musil D, Frech M, and Gerwert K
- Subjects
- Crystallography, X-Ray, HSP90 Heat-Shock Proteins chemistry, HSP90 Heat-Shock Proteins metabolism, Humans, Ligands, Models, Molecular, Molecular Conformation, Pyrazoles chemistry, Spectroscopy, Fourier Transform Infrared, Time Factors, Triazoles chemistry, Drug Discovery, HSP90 Heat-Shock Proteins antagonists & inhibitors, Pyrazoles pharmacology, Triazoles pharmacology
- Abstract
Investigation of protein-ligand interactions is crucial during early drug-discovery processes. ATR-FTIR spectroscopy can detect label-free protein-ligand interactions with high spatiotemporal resolution. Here we immobilized, as an example, the heat shock protein HSP90 on an ATR crystal. This protein is an important molecular target for drugs against several diseases including cancer. With our novel approach we investigated a ligand-induced secondary structural change. Two specific binding modes of 19 drug-like compounds were analyzed. Different binding modes can lead to different efficacy and specificity of different drugs. In addition, the k
obs values of ligand dissociation were obtained. The results were validated by X-ray crystallography for the structural change and by SPR experiments for the dissociation kinetics, but our method yields all data in a single and simple experiment., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)- Published
- 2018
- Full Text
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18. Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor, Neratinib, in Cancer Cells.
- Author
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Aljakouch K, Lechtonen T, Yosef HK, Hammoud MK, Alsaidi W, Kötting C, Mügge C, Kourist R, El-Mashtoly SF, and Gerwert K
- Subjects
- Cell Line, Tumor, ErbB Receptors metabolism, Humans, Receptor, ErbB-2 metabolism, Spectrum Analysis, Raman, Lysosomes metabolism, Neoplasms metabolism, Protein Kinase Inhibitors metabolism, Quinolines metabolism
- Abstract
Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label-free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC-MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)
- Published
- 2018
- Full Text
- View/download PDF
19. Highly stable protein immobilization via maleimido-thiol chemistry to monitor enzymatic activity.
- Author
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Schartner J, Güldenhaupt J, Katharina Gaßmeyer S, Rosga K, Kourist R, Gerwert K, and Kötting C
- Subjects
- Indicators and Reagents, Surface Properties, Enzymes, Immobilized metabolism, Spectroscopy, Fourier Transform Infrared, Sulfhydryl Compounds chemistry
- Abstract
Immobilizing enzymes for biocatalysis offers many advantages, including easy separation of the enzyme from the product and repeated and continuous use. ATR-FTIR spectroscopy is a versatile tool to monitor immobilized enzymes and has been applied to many proteins. However, while the common and convenient immobilization via oligohistidine on mono-NTA layers is adequate for the measurement of difference spectra induced by ligand binding or photochemistry, it lacks the long term stability that is necessary for monitoring biocatalysis. Here, we report a new immobilization methodology based on maleimido-thiol chemistry. A 12-mercaptododecanoic acid NHS ester monolayer is reacted with 1-(2-aminoethyl)-maleimide to build a thiol reactive surface. Subsequently, NTA-C16-thiol is covalently attached and finally oligohistidine tagged enzymes were immobilized to this surface, which remained bound with a five times higher EC50-value compared to typical mono-NTA layers. To demonstrate the high potential of the surface we analysed decarboxylation reactions catalyzed by arylmalonate decarboxylase. With ATR-FTIR both the enzyme and its substrate conversion can be monitored label free. Correct folding of the enzyme can be evaluated based on the amide band of the immobilized enzyme. In addition, the infrared absorption spectra of educt and product are monitored in real time. We show that hybrid hard-soft multivariate curve resolution improves separation of the product and educt spectra from other effects during the experiments, leading to clean kinetic traces and reaction rates for the catalytic process. Our approach can in principle be extended to any enzyme and is ideally suited for the development of biocatalysts.
- Published
- 2018
- Full Text
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20. The protonation states of GTP and GppNHp in Ras proteins.
- Author
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Mann D, Güldenhaupt J, Schartner J, Gerwert K, and Kötting C
- Subjects
- Crystallography, X-Ray, Humans, Hydrogenation, Hydrolysis, Molecular Dynamics Simulation, Guanosine Triphosphate chemistry, Guanylyl Imidodiphosphate chemistry, Protons, ras Proteins chemistry
- Abstract
The small GTPase Ras transmits signals in a variety of cellular signaling pathways, most prominently in cell proliferation. GTP hydrolysis in the active center of Ras acts as a prototype for many GTPases and is the key to the understanding of several diseases, including cancer. Therefore, Ras has been the focus of intense research over the last decades. A recent neutron diffraction crystal structure of Ras indicated a protonated γ-guanylyl imidodiphosphate (γ-GppNHp) group, which has put the protonation state of GTP in question. A possible protonation of GTP was not considered in previously published mechanistic studies. To determine the detailed prehydrolysis state of Ras, we calculated infrared and NMR spectra from quantum mechanics/molecular mechanics (QM/MM) simulations and compared them with those from previous studies. Furthermore, we measured infrared spectra of GTP and several GTP analogs bound to lipidated Ras on a membrane system under near-native conditions. Our findings unify results from previous studies and indicate a structural model confirming the hypothesis that γ-GTP is fully deprotonated in the prehydrolysis state of Ras., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2018
- Full Text
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21. Specific Substates of Ras To Interact with GAPs and Effectors: Revealed by Theoretical Simulations and FTIR Experiments.
- Author
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Li Y, Zhang Y, Großerüschkamp F, Stephan S, Cui Q, Kötting C, Xia F, and Gerwert K
- Abstract
The oncogenic Ras protein adopts various specific conformational states to execute its function in signal transduction. The large number of Ras structures obtained from X-ray and NMR experiments illustrates the diverse conformations that Ras adopts. It is difficult, however, to connect specific structural features with Ras functions. We report the free-energy landscape of Ras·GTP based on extensive explicit solvent simulations. The free-energy map clearly shows that the functional state 2 of Ras·GTP in fact has two distinct substates, denoted here as "Tyr32
in " and "Tyr32out ". Unbiased MD simulations show that the two substrates interconvert on the submicrosecond scale in solution, pointing to a novel mechanism for Ras·GTP to selectively interact with GAPs and effectors. This proposal is further supported by time-resolved FTIR experiments, which demonstrate that Tyr32 destabilizes the Ras·GAP complex and facilitates an efficient termination of Ras signaling.- Published
- 2018
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22. An ATR-FTIR Sensor Unraveling the Drug Intervention of Methylene Blue, Congo Red, and Berberine on Human Tau and Aβ.
- Author
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Schartner J, Nabers A, Budde B, Lange J, Hoeck N, Wiltfang J, Kötting C, and Gerwert K
- Abstract
Alzheimer's disease affects millions of human beings worldwide. The disease progression is characterized by the formation of plaques and neurofibrillary tangles in the brain, which are based on aggregation processes of the Aβ peptide and tau protein. Today there is no cure and even no in vitro assay available for the identification of drug candidates, which provides direct information concerning the protein secondary structure label-free. Therefore, we developed an attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) sensor, which uses surface bound antibodies to immobilize a desired target protein. The secondary structure of the protein can be evaluated based on the secondary structure sensitive frequency of the amide I band. Direct information about the effect of a drug candidate on the secondary structure distribution of the total target protein fraction within the respective body fluid can be detected by a frequency shift of the amide I band. Thereby, the extent of the amide I shift is indicative for the compound efficiency. The functionality of this approach was demonstrated by the quantification of the effect of the drug candidate methylene blue on the pathogenic misfolded tau protein as extracted from cerebrospinal fluid (CSF). Methylene blue induces a shift from pathogenic folded β-sheet dominated to the healthy monomeric state. A similar effect was observed for congo red on pathogenic Aβ isoforms from CSF. In addition, the effect of berberine on synthetic Aβ
1-42 is studied. Berberine seems to decelerate the aggregation process of synthetic Aβ1-42 peptides.- Published
- 2017
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23. Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs.
- Author
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Gerwert K, Mann D, and Kötting C
- Subjects
- Biocatalysis, Cell Membrane metabolism, Guanosine Triphosphate metabolism, GTP Phosphohydrolases chemistry, GTP Phosphohydrolases metabolism, GTPase-Activating Proteins metabolism, Protein Multimerization
- Abstract
GTPases are central switches in cells. Their dysfunctions are involved in severe diseases. The small GTPase Ras regulates cell growth, differentiation and apoptosis by transmitting external signals to the nucleus. In one group of oncogenic mutations, the 'switch-off' reaction is inhibited, leading to persistent activation of the signaling pathway. The switch reaction is regulated by GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis in Ras, and by guanine nucleotide exchange factors, which catalyze the exchange of GDP for GTP. Heterotrimeric G-proteins are activated by G-protein coupled receptors and are inactivated by GTP hydrolysis in the Gα subunit. Their GAPs are called regulators of G-protein signaling. In the same way that Ras serves as a prototype for small GTPases, Gαi1 is the most well-studied Gα subunit. By utilizing X-ray structural models, time-resolved infrared-difference spectroscopy, and biomolecular simulations, we elucidated the detailed molecular reaction mechanism of the GTP hydrolysis in Ras and Gαi1. In both proteins, the charge distribution of GTP is driven towards the transition state, and an arginine is precisely positioned to facilitate nucleophilic attack of water. In addition to these mechanistic details of GTP hydrolysis, Ras dimerization as an emerging factor in signal transduction is discussed in this review.
- Published
- 2017
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24. Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy.
- Author
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Mann D, Höweler U, Kötting C, and Gerwert K
- Subjects
- Amino Acid Motifs, GTP-Binding Protein alpha Subunits, Gi-Go genetics, Guanosine Diphosphate metabolism, Hydrogen Bonding, Hydrolysis, Magnesium metabolism, Mutation, Quantum Theory, GTP-Binding Protein alpha Subunits, Gi-Go chemistry, Molecular Dynamics Simulation, Spectroscopy, Fourier Transform Infrared
- Abstract
Time-resolved Fourier transform infrared (FTIR) spectroscopy is a powerful tool to elucidate label-free the reaction mechanisms of proteins. After assignment of the absorption bands to individual groups of the protein, the order of events during the reaction mechanism can be monitored and rate constants can be obtained. Additionally, structural information is encoded into infrared spectra and can be decoded by combining the experimental data with biomolecular simulations. We have determined recently the infrared vibrations of GTP and guanosine diphosphate (GDP) bound to Gα
i1 , a ubiquitous GTPase. These vibrations are highly sensitive for the environment of the phosphate groups and thereby for the binding mode the GTPase adopts to enable fast hydrolysis of GTP. In this study we calculated these infrared vibrations from biomolecular simulations to transfer the spectral information into a computational model that provides structural information far beyond crystal structure resolution. Conformational ensembles were generated using 15 snapshots of several 100 ns molecular-mechanics/molecular-dynamics (MM-MD) simulations, followed by quantum-mechanics/molecular-mechanics (QM/MM) minimization and normal mode analysis. In comparison with other approaches, no time-consuming QM/MM-MD simulation was necessary. We carefully benchmarked the simulation systems by deletion of single hydrogen bonds between the GTPase and GTP through several Gαi1 point mutants. The missing hydrogen bonds lead to blue-shifts of the corresponding absorption bands. These band shifts for α-GTP (Gαi1 -T48A), γ-GTP (Gαi1 -R178S), and for both β-GTP/γ-GTP (Gαi1 -K46A, Gαi1 -D200E) were found in agreement in the experimental and the theoretical spectra. We applied our approach to open questions regarding Gαi1 : we show that the GDP state of Gαi1 carries a Mg2+ , which is not found in x-ray structures. Further, the catalytic role of K46, a central residue of the P-loop, and the protonation state of the GTP are elucidated., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2017
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25. Mechanism of the intrinsic arginine finger in heterotrimeric G proteins.
- Author
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Mann D, Teuber C, Tennigkeit SA, Schröter G, Gerwert K, and Kötting C
- Subjects
- Arginine chemistry, Catalytic Domain, Enzyme Stability, GTP-Binding Protein alpha Subunits, Gi-Go chemistry, GTP-Binding Protein alpha Subunits, Gi-Go genetics, GTP-Binding Protein alpha Subunits, Gi-Go metabolism, Guanosine Triphosphate chemistry, Guanosine Triphosphate metabolism, Heterotrimeric GTP-Binding Proteins genetics, Heterotrimeric GTP-Binding Proteins metabolism, Humans, Hydrolysis, Models, Molecular, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Neurofibromin 1 chemistry, Neurofibromin 1 metabolism, RGS Proteins chemistry, RGS Proteins genetics, RGS Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Spectroscopy, Fourier Transform Infrared, Heterotrimeric GTP-Binding Proteins chemistry
- Abstract
Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gα
i1 , which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1 ·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis., Competing Interests: The authors declare no conflict of interest.- Published
- 2016
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26. Unraveling the Phosphocholination Mechanism of the Legionella pneumophila Enzyme AnkX.
- Author
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Gavriljuk K, Schartner J, Seidel H, Dickhut C, Zahedi RP, Hedberg C, Kötting C, and Gerwert K
- Subjects
- Ankyrin Repeat, Bacterial Proteins genetics, Biocatalysis, Catalytic Domain, Diacylglycerol Cholinephosphotransferase genetics, Host-Pathogen Interactions, Humans, Legionella pneumophila genetics, Legionella pneumophila pathogenicity, Models, Molecular, Phosphorylcholine metabolism, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Spectroscopy, Fourier Transform Infrared, rab GTP-Binding Proteins metabolism, rab1 GTP-Binding Proteins metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Diacylglycerol Cholinephosphotransferase chemistry, Diacylglycerol Cholinephosphotransferase metabolism, Legionella pneumophila enzymology
- Abstract
The intracellular pathogen Legionella pneumophila infects lung macrophages and injects numerous effector proteins into the host cell to establish a vacuole for proliferation. The necessary interference with vesicular trafficking of the host is achieved by modulation of the function of Rab GTPases. The effector protein AnkX chemically modifies Rab1b and Rab35 by covalent phosphocholination of serine or threonine residues using CDP-choline as a donor. So far, the phosphoryl transfer mechanism and the relevance of observed autophosphocholination of AnkX remained disputable. We designed tailored caged compounds to make this type of enzymatic reaction accessible for time-resolved Fourier transform infrared difference spectroscopy. By combining spectroscopic and biochemical methods, we determined that full length AnkX is autophosphocholinated at Ser521, Thr620, and Thr943. However, autophosphocholination loses specificity for these sites in shortened constructs and does not appear to be relevant for the catalysis of the phosphoryl transfer. In contrast, transient phosphocholination of His229 in the conserved catalytic motif might exist as a short-lived reaction intermediate. Upon substrate binding, His229 is deprotonated and locked in this state, being rendered capable of a nucleophilic attack on the pyrophosphate moiety of the substrate. The proton that originated from His229 is transferred to a nearby carboxylic acid residue. Thus, our combined findings support a ping-pong mechanism involving phosphocholination of His229 and subsequent transfer of phosphocholine to the Rab GTPase. Our approach can be extended to the investigation of further nucleotidyl transfer reactions, which are currently of reemerging interest in regulatory pathways of host-pathogen interactions.
- Published
- 2016
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27. Amyloid-β-Secondary Structure Distribution in Cerebrospinal Fluid and Blood Measured by an Immuno-Infrared-Sensor: A Biomarker Candidate for Alzheimer's Disease.
- Author
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Nabers A, Ollesch J, Schartner J, Kötting C, Genius J, Hafermann H, Klafki H, Gerwert K, and Wiltfang J
- Subjects
- Aged, Amyloid beta-Peptides blood, Amyloid beta-Peptides cerebrospinal fluid, Female, Humans, Male, Middle Aged, Prospective Studies, Protein Structure, Secondary, Alzheimer Disease blood, Alzheimer Disease cerebrospinal fluid, Amyloid beta-Peptides chemistry, Biomarkers blood, Biomarkers cerebrospinal fluid
- Abstract
The misfolding of the Amyloid-beta (Aβ) peptide into β-sheet enriched conformations was proposed as an early event in Alzheimer's Disease (AD). Here, the Aβ peptide secondary structure distribution in cerebrospinal fluid (CSF) and blood plasma of 141 patients was measured with an immuno-infrared-sensor. The sensor detected the amide I band, which reflects the overall secondary structure distribution of all Aβ peptides extracted from the body fluid. We observed a significant downshift of the amide I band frequency of Aβ peptides in Dementia Alzheimer type (DAT) patients, which indicated an overall shift to β-sheet. The secondary structure distribution of all Aβ peptides provides a better marker for DAT detection than a single Aβ misfold or the concentration of a specific oligomer. The discrimination between DAT and disease control patients according to the amide I frequency was in excellent agreement with the clinical diagnosis (accuracy 90% for CSF and 84% for blood). The amide I band maximum above or below the decisive marker frequency appears as a novel spectral biomarker candidate of AD. Additionally, a preliminary proof-of-concept study indicated an amide I band shift below the marker band already in patients with mild cognitive impairment due to AD. The presented immuno-IR-sensor method represents a promising, simple, robust, and label-free diagnostic tool for CSF and blood analysis.
- Published
- 2016
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28. An infrared sensor analysing label-free the secondary structure of the Abeta peptide in presence of complex fluids.
- Author
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Nabers A, Ollesch J, Schartner J, Kötting C, Genius J, Haußmann U, Klafki H, Wiltfang J, and Gerwert K
- Subjects
- Animals, Biomimetics, Chick Embryo, Models, Molecular, Protein Structure, Secondary, Amyloid beta-Peptides chemistry, Spectroscopy, Fourier Transform Infrared instrumentation, Water chemistry
- Abstract
The secondary structure change of the Abeta peptide to beta-sheet was proposed as an early event in Alzheimer's disease. The transition may be used for diagnostics of this disease in an early state. We present an Attenuated Total Reflection (ATR) sensor modified with a specific antibody to extract minute amounts of Abeta peptide out of a complex fluid. Thereby, the Abeta peptide secondary structure was determined in its physiological aqueous environment by FTIR-difference-spectroscopy. The presented results open the door for label-free Alzheimer diagnostics in cerebrospinal fluid or blood. It can be extended to further neurodegenerative diseases. An immunologic ATR-FTIR sensor for Abeta peptide secondary structure analysis in complex fluids is presented., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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29. Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental, and theoretical IR spectroscopy.
- Author
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Rudack T, Jenrich S, Brucker S, Vetter IR, Gerwert K, and Kötting C
- Subjects
- Catalysis, Catalytic Domain, Crystallography, X-Ray, Humans, Hydrolysis, Magnesium chemistry, Manganese chemistry, Molecular Dynamics Simulation, Mutation, Phosphates chemistry, Protein Binding, Spectroscopy, Fourier Transform Infrared, Tyrosine chemistry, GTP Phosphohydrolases chemistry, GTPase-Activating Proteins chemistry, Guanosine Triphosphate chemistry, Membrane Proteins chemistry, Spectrophotometry, Infrared
- Abstract
Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg(2+) coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg(2+) in GTPases. The Mg(2+) coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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30. Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells.
- Author
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El-Mashtoly SF, Yosef HK, Petersen D, Mavarani L, Maghnouj A, Hahn S, Kötting C, and Gerwert K
- Subjects
- Antibodies, Monoclonal chemistry, Antineoplastic Agents chemistry, ErbB Receptors chemistry, Humans, Multivariate Analysis, Mutation, Panitumumab, Spectrum Analysis, Raman, Structure-Activity Relationship, Tumor Cells, Cultured, ras Proteins genetics, Antibodies, Monoclonal pharmacology, Antineoplastic Agents pharmacology, ErbB Receptors antagonists & inhibitors
- Abstract
Predictions about the cellular efficacy of drugs tested in vitro are usually based on the measured responses of a few proteins or signal transduction pathways. However, cellular proteins are highly coupled in networks, and observations of single proteins may not adequately reflect the in vivo cellular response to drugs. This might explain some large discrepancies between in vitro drug studies and drug responses observed in patients. We present a novel in vitro marker-free approach that enables detection of cellular responses to a drug. We use Raman spectral imaging to measure the effect of the epidermal growth factor receptor (EGFR) inhibitor panitumumab on cell lines expressing wild-type Kirsten-Ras (K-Ras) and oncogenic K-Ras mutations. Oncogenic K-Ras mutation blocks the response to anti-EGFR therapy in patients, but this effect is not readily observed in vitro. The Raman studies detect large panitumumab-induced differences in vitro in cells harboring wild-type K-Ras as seen in A in red but not in cells with K-Ras mutations as seen in B; these studies reflect the observed patient outcomes. However, the effect is not observed when extracellular-signal-regulated kinase phosphorylation is monitored. The Raman spectra show for cells with wild-type K-Ras alterations based on the responses to panitumumab. The subcellular component with the largest spectral response to panitumumab was lipid droplets, but this effect was not observed when cells harbored K-Ras mutations. This study develops a noninvasive, label-free, in vitro vibrational spectroscopic test to determine the integral physiologically relevant drug response in cell lines. This approach opens a new field of patient-centered drug testing that could deliver superior patient therapies.
- Published
- 2015
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31. Chemical Functionalization of Germanium with Dextran Brushes for Immobilization of Proteins Revealed by Attenuated Total Reflection Fourier Transform Infrared Difference Spectroscopy.
- Author
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Schartner J, Hoeck N, Güldenhaupt J, Mavarani L, Nabers A, Gerwert K, and Kötting C
- Subjects
- Dextrans chemistry, Germanium isolation & purification, Microscopy, Atomic Force, Models, Molecular, Molecular Structure, Particle Size, Spectroscopy, Fourier Transform Infrared, Surface Properties, Red Fluorescent Protein, Chemical Fractionation, Dextrans isolation & purification, Germanium chemistry, Green Fluorescent Proteins chemistry, Immobilized Proteins chemistry, Luminescent Proteins chemistry
- Abstract
Protein immobilization studied by attenuated total reflection Fourier transform infrared (ATR-FT-IR) difference spectroscopy is an emerging field enabling the study of proteins at atomic detail. Gold or glass surfaces are frequently used for protein immobilization. Here, we present an alternative method for protein immobilization on germanium. Because of its high refractive index and broad spectral window germanium is the best material for ATR-FT-IR spectroscopy of thin layers. So far, this technique was mainly used for protein monolayers, which lead to a limited signal-to-noise ratio. Further, undesired protein-protein interactions can occur in a dense layer. Here, the germanium surface was functionalized with thiols and stepwise a dextran brush was generated. Each step was monitored by ATR-FT-IR spectroscopy. We compared a 70 kDa dextran with a 500 kDa dextran regarding the binding properties. All surfaces were characterized by atomic force microscopy, revealing thicknesses between 40 and 110 nm. To analyze the capability of our system we utilized N-Ras on mono-NTA (nitrilotriacetic acid) functionalized dextran, and the amount of immobilized Ras corresponded to several monolayers. The protein stability and loading capacity was further improved by means of tris-NTA for immobilization. Small-molecule-induced changes were revealed with an over 3 times higher signal-to-noise ratio compared to monolayers. This improvement may allow the observation of very small and so far hidden changes in proteins upon stimulus. Furthermore, we immobilized green fluorescent protein (GFP) and mCherry simultaneously enabling an analysis of the surface by fluorescence microscopy. The absence of a Förster resonance energy transfer (FRET) signal demonstrated a large protein-protein distance, indicating an even distribution of the protein within the dextran.
- Published
- 2015
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32. Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism.
- Author
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Schröter G, Mann D, Kötting C, and Gerwert K
- Subjects
- GTP-Binding Protein alpha Subunits, Gi-Go genetics, Guanosine Diphosphate metabolism, Guanosine Triphosphate metabolism, Humans, Hydrolysis, Kinetics, Models, Molecular, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Protein Conformation, Protein Interaction Domains and Motifs, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Spectrometry, Fluorescence, Spectroscopy, Fourier Transform Infrared, GTP-Binding Protein alpha Subunits, Gi-Go chemistry, GTP-Binding Protein alpha Subunits, Gi-Go metabolism
- Abstract
Gα subunits are central molecular switches in cells. They are activated by G protein-coupled receptors that exchange GDP for GTP, similar to small GTPase activation mechanisms. Gα subunits are turned off by GTP hydrolysis. For the first time we employed time-resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of Gαi1. FTIR spectroscopy is a powerful tool that monitors reactions label free with high spatio-temporal resolution. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange/Mg(2+) binding bias. Global fit analysis resulted in one apparent rate constant of 0.02 s(-1) at 15 °C. Isotopic labeling was applied to assign the individual phosphate vibrations for α-, β-, and γ-GTP (1243, 1224, and 1156 cm(-1), respectively), α- and β-GDP (1214 and 1134/1103 cm(-1), respectively), and free phosphate (1078/991 cm(-1)). In contrast to Ras · GAP catalysis, the bond breakage of the β-γ-phosphate but not the Pi release is rate-limiting in the GTPase reaction. Complementary common GTPase assays were used. Reversed phase HPLC provided multiple turnover rates and tryptophan fluorescence provided nucleotide exchange rates. Experiments were complemented by molecular dynamics simulations. This broad approach provided detailed insights at atomic resolution and allows now to identify key residues of Gαi1 in GTP hydrolysis and nucleotide exchange. Mutants of the intrinsic arginine finger (Gαi1-R178S) affected exclusively the hydrolysis reaction. The effect of nucleotide binding (Gαi1-D272N) and Ras-like/all-α interface coordination (Gαi1-D229N/Gαi1-D231N) on the nucleotide exchange reaction was furthermore elucidated., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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33. What vibrations tell us about GTPases.
- Author
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Kötting C and Gerwert K
- Subjects
- Humans, Models, Molecular, Vibration, GTP Phosphohydrolases metabolism, Spectroscopy, Fourier Transform Infrared methods
- Abstract
In this review, we discuss how time-resolved Fourier transform infrared (FTIR) spectroscopy is used to understand how GTP hydrolysis is catalyzed by small GTPases and their cognate GTPase-activating proteins (GAPs). By interaction with small GTPases, GAPs regulate important signal transduction pathways and transport mechanisms in cells. The GTPase reaction terminates signaling and controls transport. Dysfunctions of GTP hydrolysis in these proteins are linked to serious diseases including cancer. Using FTIR, we resolved both the intrinsic and GAP-catalyzed GTPase reaction of the small GTPase Ras with high spatiotemporal resolution and atomic detail. This provided detailed insight into the order of events and how the active site is completed for catalysis. Comparisons of Ras with other small GTPases revealed conservation and variation in the catalytic mechanisms. The approach was extended to more nearly physiological conditions at a membrane. Interactions of membrane-anchored GTPases and their extraction from the membrane are studied using the attenuated total reflection (ATR) technique.
- Published
- 2015
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34. Immobilization of proteins in their physiological active state at functionalized thiol monolayers on ATR-germanium crystals.
- Author
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Schartner J, Gavriljuk K, Nabers A, Weide P, Muhler M, Gerwert K, and Kötting C
- Subjects
- Histidine chemistry, Histidine genetics, Histidine metabolism, Models, Molecular, Molecular Structure, Rhodopsin chemistry, Rhodopsin metabolism, Spectroscopy, Fourier Transform Infrared, Germanium chemistry, Immobilized Proteins chemistry, Immobilized Proteins metabolism, Sulfhydryl Compounds chemistry
- Abstract
Protein immobilization on solid surfaces has become a powerful tool for the investigation of protein function. Physiologically relevant molecular reaction mechanisms and interactions of proteins can be revealed with excellent signal-to-noise ratio by vibrational spectroscopy (ATR-FTIR) on germanium crystals. Protein immobilization by thiol chemistry is well-established on gold surfaces, for example, for surface plasmon resonance. Here, we combine features of both approaches: a germanium surface functionalized with different thiols to allow specific immobilization of various histidine-tagged proteins with over 99% specific binding. In addition to FTIR, the surfaces were characterized by XPS and fluorescence microscopy. Secondary-structure analysis and stimulus-induced difference spectroscopy confirmed protein activity at the atomic level, for example, physiological cation channel formation of Channelrhodopsin 2., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
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35. Reaction mechanism of adenylyltransferase DrrA from Legionella pneumophila elucidated by time-resolved fourier transform infrared spectroscopy.
- Author
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Gavriljuk K, Schartner J, Itzen A, Goody RS, Gerwert K, and Kötting C
- Subjects
- Adenosine Monophosphate metabolism, Adenosine Triphosphate metabolism, Bacterial Proteins genetics, Binding Sites, Catalytic Domain, Guanine Nucleotide Exchange Factors genetics, Kinetics, Models, Molecular, Mutagenesis, Site-Directed, Phosphates chemistry, Protein Conformation, Spectroscopy, Fourier Transform Infrared, Tyrosine metabolism, rab1 GTP-Binding Proteins metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Guanine Nucleotide Exchange Factors chemistry, Guanine Nucleotide Exchange Factors metabolism
- Abstract
Modulation of the function of small GTPases that regulate vesicular trafficking is a strategy employed by several human pathogens. Legionella pneumophila infects lung macrophages and injects a plethora of different proteins into its host cell. Among these is DrrA/SidM, which catalyzes stable adenylylation of Rab1b, a regulator of endoplasmatic reticulum to Golgi trafficking, and thereby alters the function and interactions of this small GTPase. We employed time-resolved FTIR-spectroscopy to monitor the DrrA-catalyzed AMP-transfer to Tyr77 of Rab1b. A transient complex between DrrA, adenylylated Rab1b, and the pyrophosphate byproduct was resolved, allowing us to analyze the interactions at the active site. Combination of isotopic labeling and site-directed mutagenesis allowed us to derive the catalytic mechanism of DrrA from the FTIR difference spectra. DrrA shares crucial residues in the ATP-binding pocket with similar AMP-transferring enzymes such as glutamine synthetase adenylyltransferase or kanamycin nucleotidyltransferase, but provides the complete active site on a single subunit. We determined that Asp112 of DrrA functions as the catalytic base for deprotonation of Tyr77 of Rab1b to enable nucleophilic attack on the ATP. The study provides detailed understanding of the Legionella pneumophila protein DrrA and of AMP-transfer reactions in general.
- Published
- 2014
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36. Automated identification of subcellular organelles by coherent anti-stokes Raman scattering.
- Author
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El-Mashtoly SF, Niedieker D, Petersen D, Krauss SD, Freier E, Maghnouj A, Mosig A, Hahn S, Kötting C, and Gerwert K
- Subjects
- Automation, Cell Line, Tumor, Cluster Analysis, Feasibility Studies, Humans, Pancreatic Neoplasms pathology, Molecular Imaging methods, Organelles metabolism, Spectrum Analysis, Raman methods
- Abstract
Coherent anti-Stokes Raman scattering (CARS) is an emerging tool for label-free characterization of living cells. Here, unsupervised multivariate analysis of CARS datasets was used to visualize the subcellular compartments. In addition, a supervised learning algorithm based on the "random forest" ensemble learning method as a classifier, was trained with CARS spectra using immunofluorescence images as a reference. The supervised classifier was then used, to our knowledge for the first time, to automatically identify lipid droplets, nucleus, nucleoli, and endoplasmic reticulum in datasets that are not used for training. These four subcellular components were simultaneously and label-free monitored instead of using several fluorescent labels. These results open new avenues for label-free time-resolved investigation of subcellular components in different cells, especially cancer cells., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
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37. Label-free imaging of drug distribution and metabolism in colon cancer cells by Raman microscopy.
- Author
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El-Mashtoly SF, Petersen D, Yosef HK, Mosig A, Reinacher-Schick A, Kötting C, and Gerwert K
- Subjects
- Cell Line, Tumor, Colonic Neoplasms drug therapy, Colonic Neoplasms pathology, Erlotinib Hydrochloride, Humans, Microscopy, Confocal methods, Quinazolines therapeutic use, Colonic Neoplasms metabolism, Quinazolines metabolism, Spectrum Analysis, Raman methods
- Abstract
Targeted cancer therapies block cancer growth and spread using small molecules. Many molecular targets for an epidermal growth factor receptor (EGFR) selectively compete with the adenosine triphosphate-binding site of its tyrosine kinase domain. Detection of molecular targeted agents and their metabolites in cells/tissues by label-free imaging is attractive because dyes or fluorescent labels may be toxic or invasive. Here, label-free Raman microscopy is applied to show the spatial distribution of the molecular targeted drug erlotinib within the cell. The Raman images show that the drug is clustered at the EGFR protein at the membrane and induces receptor internalization. The changes within the Raman spectrum of erlotinib measured in cells as compared to the free-erlotinib spectrum indicate that erlotinib is metabolized within cells to its demethylated derivative. This study provides detailed insights into the drug targeting mechanism at the atomic level in cells. It demonstrates that Raman microscopy will open avenues as a non-invasive and label-free technique to investigate pharmacokinetics at the highest possible resolution in living cells.
- Published
- 2014
- Full Text
- View/download PDF
38. A modified infrared spectrometer with high time resolution and its application for investigating fast conformational changes of the GTPase Ras.
- Author
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Lin J, Gerwert K, and Kötting C
- Subjects
- Binding Sites, Guanosine Triphosphate chemistry, Guanosine Triphosphate radiation effects, Lasers, Semiconductor, Models, Molecular, Photolysis, Protein Binding, Protein Conformation, Protein Interaction Mapping, Protein Structure, Tertiary, Proto-Oncogene Proteins p21(ras) metabolism, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Spectrophotometry, Infrared statistics & numerical data, Temperature, Thermodynamics, Time, raf Kinases metabolism, Guanosine Triphosphate analogs & derivatives, Proto-Oncogene Proteins p21(ras) chemistry, Spectrophotometry, Infrared instrumentation, raf Kinases chemistry
- Abstract
Time-resolved infrared spectroscopy is a valuable tool for the investigation of proteins and protein interactions. The investigation of many biological processes is possible by means of caged compounds, which set free biologically active substances upon light activation. Some caged compounds could provide sub-nanosecond time resolution, e.g., para-hydroxyphenacyl-guanosine 5'-triphosphate (GTP) forms GTP in picoseconds. However, the time resolution in single shot experiments with rapid-scan Fourier transform infrared (FT-IR) spectrometers is limited to about 10 ms. Here we use an infrared diode laser instead of the conventional globar and achieve a time resolution of 100 ns. This allows for the time-resolved measurement of the fast Ras(off) to Ras(on) conformational change at room temperature. We quantified the activation parameters for this reaction and found that the free energy of activation for this reaction is mainly enthalpic. Investigation of the same reaction in the presence of the Ras binding domain of the effector Raf (RafRBD) reveals a four orders of magnitude faster reaction, indicating that Ras·RafRBD complex formation directly induces the conformational change. Recent developments of broadly tunable quantum cascade lasers will further improve time resolution and usability of the setup. The reported 100 ns time resolution is the best achieved for a non-repetitive experiment so far.
- Published
- 2014
- Full Text
- View/download PDF
39. Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy.
- Author
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Gavriljuk K, Itzen A, Goody RS, Gerwert K, and Kötting C
- Subjects
- Animals, Cattle, Kinetics, Phosphorylcholine metabolism, Prenylation, Saccharomyces cerevisiae, Spectroscopy, Fourier Transform Infrared methods, Guanine Nucleotide Dissociation Inhibitors metabolism, Legionella pneumophila metabolism, Membranes metabolism, Spectrophotometry, Infrared methods, Transport Vesicles metabolism, rab GTP-Binding Proteins metabolism
- Abstract
Membrane trafficking is regulated by small Ras-like GDP/GTP binding proteins of the Rab subfamily (Rab GTPases) that cycle between membranes and cytosol depending on their nucleotide state. The GDP dissociation inhibitor (GDI) solubilizes prenylated Rab GTPases from and shuttles them between membranes in the form of a soluble cytosolic complex. We use attenuated total reflection-Fourier transform infrared spectroscopy to directly observe extraction of Rab GTPases from model membranes by GDI. In their native form, most Rab GTPases are doubly geranylgeranylated at the C terminus to achieve localization to the membrane. We find that monogeranylgeranylated Rab35 and Rab1b reversibly bind to a negatively charged model membrane. Correct folding and GTPase activity of the membrane-bound protein can be evaluated. The dissociation kinetics depends on the C-terminal sequence and charge of the GTPases. The attenuated total reflection experiments show that GDI genuinely accelerates the intrinsic Rab membrane dissociation. The extraction process is characterized and occurs in a nucleotide-dependent manner. Furthermore, we find that phosphocholination of Rab35, which is catalyzed by the Legionella pneumophila protein AnkX, interferes with the ability of GDI to extract Rab35 from the membrane. The attenuated total reflection-Fourier transform infrared spectroscopy approach enables label-free investigation of the interaction between GDI and Rab GTPases in a membrane environment. Thereby, GDI is revealed to actively extract monogeranylgeranylated membrane-bound Rab GTPases and, thus, is not merely a solubilization factor.
- Published
- 2013
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40. Spectral histopathology of colon cancer tissue sections by Raman imaging with 532 nm excitation provides label free annotation of lymphocytes, erythrocytes and proliferating nuclei of cancer cells.
- Author
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Mavarani L, Petersen D, El-Mashtoly SF, Mosig A, Tannapfel A, Kötting C, and Gerwert K
- Subjects
- Cell Nucleus genetics, Colonic Neoplasms genetics, Colonic Neoplasms surgery, Fluorescence, Humans, Immunoenzyme Techniques, Mutation genetics, Spectroscopy, Fourier Transform Infrared, Tumor Cells, Cultured, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism, Cell Nucleus pathology, Cell Proliferation, Colonic Neoplasms diagnosis, Diagnostic Imaging, Erythrocytes pathology, Lymphocytes pathology, Spectrum Analysis, Raman methods
- Abstract
Spectral histopathology (SHP) is an emerging tool for label free annotation of tissue. While FTIR based SHP provides fast annotation of larger tissue sections, Raman based SHP is slower but achieves a 10 times higher spatial resolution as compared to FTIR. Usually NIR excitation is used for Raman measurements on biological samples. Here, for the first time 532 nm excitation is used to annotate colon tissue by Raman SHP. Excellent data quality is obtained, which resolves for example erythrocytes and lymphocytes. In addition to Raman scattering auto-fluorescence is observed. We found that this auto-fluorescence overlaps spatially with the fluorescence of antibodies against p53 used in routine immunohistochemistry in surgical pathology. This fluorescence indicates nuclei of cancer cells with mutated p53 and allows new label free assignment of cancer cells. These results open new avenues for optical diagnosis by Raman spectroscopy and autofluorescence.
- Published
- 2013
- Full Text
- View/download PDF
41. The dynamics of the catalytic site in small GTPases, variations on a common motif.
- Author
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Kötting C and Gerwert K
- Subjects
- Amino Acid Motifs, Catalytic Domain, Kinetics, Protein Binding, Spectroscopy, Fourier Transform Infrared, Models, Molecular, Monomeric GTP-Binding Proteins chemistry
- Abstract
Small GTPases control many cellular processes. Their catalytic downregulation by GTPase activating proteins (GAP) is essential. Many structural models of GTPase·GAP complexes obtained by X-ray structural analysis are available nowadays. They reveal important insights into the catalytic site and can suggest important catalytic residues. But this information is static. Time-resolved FTIR spectroscopy can resolve the dynamics of the catalytic site at atomic detail. For the investigation of GAP catalyzed GTPase reactions of small GTPases, the order of events like the action of certain catalytic amino acids, bond breakages and protein conformational changes can be elucidated. This is elaborated for many small GTPases like Ras, Rap, Ran, Rho and Rab and their cognate GAPs. Variations on a common dynamic motif of the catalytic site of small GTPase will be presented., (Copyright © 2013. Published by Elsevier B.V.)
- Published
- 2013
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42. Universal method for protein immobilization on chemically functionalized germanium investigated by ATR-FTIR difference spectroscopy.
- Author
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Schartner J, Güldenhaupt J, Mei B, Rögner M, Muhler M, Gerwert K, and Kötting C
- Subjects
- Models, Molecular, Molecular Structure, Proteins analysis, Spectroscopy, Fourier Transform Infrared, Surface Properties, Germanium chemistry, Proteins chemistry
- Abstract
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy allows a detailed analysis of surface attached molecules, including their secondary structure, orientation, and interaction with small molecules in the case of proteins. Here, we present a universal immobilization technique on germanium for all oligo-histidine-tagged proteins. For this purpose, new triethoxysilane derivates were developed: we synthesized a linker-silane with a succinimidyl ester as amine-reactive headgroup and a matrix-silane with an unreactive ethylene glycol group. A new methodology for the attachment of triethoxysilanes on germanium was established, and the surface was characterized by ATR-FTIR and X-ray photoelectron spectroscopy. In the next step, the succinimidyl ester was reacted with aminonitrilotriacetic acid. Subsequently, Ni(2+) was coordinated to form Ni-nitrilotriacetic acid for His-tag binding. The capability of the functionalized surface was demonstrated by experiments using the small GTPase Ras and photosystem I (PS I). The native binding of the proteins was proven by difference spectroscopy, which probes protein function. The function of Ras as molecular switch was demonstrated by a beryllium trifluoride anion titration assay, which allows observation of the "on" and "off" switching of Ras at atomic resolution. Furthermore, the activity of immobilized PS I was proven by light-induced difference spectroscopy. Subsequent treatment with imidazole removes attached proteins, enabling repeated binding. This universal technique allows specific attachment of His-tagged proteins and a detailed study of their function at the atomic level using FTIR difference spectroscopy.
- Published
- 2013
- Full Text
- View/download PDF
43. Monitoring protein-ligand interactions by time-resolved FTIR difference spectroscopy.
- Author
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Kötting C and Gerwert K
- Subjects
- Archaea chemistry, Binding Sites, Guanosine Triphosphate chemistry, Humans, Hydrogen Bonding, Isotope Labeling, Kinetics, Ligands, Microfluidic Analytical Techniques, Mutagenesis, Site-Directed, Photolysis, Protein Binding, Time Factors, ras Proteins genetics, Bacteriorhodopsins chemistry, Guanosine Triphosphate analogs & derivatives, Spectroscopy, Fourier Transform Infrared methods, Thionucleotides chemistry, ras Proteins chemistry
- Abstract
Time-resolved FTIR difference spectroscopy is a valuable tool to monitor the dynamics and exact molecular details of protein-ligand interactions. FTIR difference spectroscopy selects, out of the background absorbance of the whole sample, the absorbance bands of the protein groups and of the ligands that are involved in the protein reaction. The absorbance changes can be monitored with time-resolutions down to nanoseconds and followed for time periods ranging over nine orders of magnitude even in membrane proteins with a size of 100,000 Da. Here, we discuss the various experimental setups. The rapid scan technique allows a time resolution in the millisecond regime, whereas the step scan technique allows nanosecond time resolution. We show appropriate sample cells and how to trigger a reaction within these cells. The kinetic analysis of the data is discussed. A crucial step in the data analysis is the reliable assignment of bands to chemical groups of the protein and the ligand. This is done either by site directed mutagenesis, where the absorbance bands of the exchanged amino acids disappear or by isotopically labeling, where the band of the labelled group is frequency shifted.
- Published
- 2013
- Full Text
- View/download PDF
44. Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail.
- Author
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Gavriljuk K, Gazdag EM, Itzen A, Kötting C, Goody RS, and Gerwert K
- Subjects
- Animals, Catalytic Domain, DNA Mutational Analysis, GTPase-Activating Proteins chemistry, Glutamine metabolism, Guanosine Triphosphate metabolism, Humans, Hydrolysis, Kinetics, Spectroscopy, Fourier Transform Infrared, rab1 GTP-Binding Proteins chemistry, Biocatalysis, GTPase-Activating Proteins metabolism, Mammals metabolism, Models, Molecular, rab1 GTP-Binding Proteins metabolism
- Abstract
Rab GTPases, key regulators of vesicular transport, hydrolyze GTP very slowly unless assisted by Rab GTPase-activating proteins (RabGAPs). Dysfunction of RabGAPs is involved in many diseases. By combining X-ray structure analysis and time-resolved FTIR spectroscopy we reveal here the detailed molecular reaction mechanism of a complex between human Rab and RabGAP at the highest possible spatiotemporal resolution and in atomic detail. A glutamine residue of Rab proteins (cis-glutamine) that is essential for intrinsic activity is less important in the GAP-activated reaction. During generation of the RabGAP·Rab:GTP complex, there is a rapid conformational change in which the cis-glutamine is replaced by a glutamine from RabGAP (trans-glutamine); this differs from the RasGAP mechanism, where the cis-glutamine is also important for GAP catalysis. However, as in the case of Ras, a trans-arginine is also recruited to complete the active center during this conformational change. In contrast to the RasGAP mechanism, an accumulation of a state in which phosphate is bound is not observed, and bond breakage is the rate-limiting step. The movement of trans-glutamine and trans-arginine into the catalytic site and bond breakage during hydrolysis are monitored in real time. The combination of X-ray structure analysis and time-resolved FTIR spectroscopy provides detailed insight in the catalysis of human Rab GTPases.
- Published
- 2012
- Full Text
- View/download PDF
45. Detailed structure of the H2PO4(-)-guanosine diphosphate intermediate in Ras-GAP decoded from FTIR experiments by biomolecular simulations.
- Author
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Xia F, Rudack T, Cui Q, Kötting C, and Gerwert K
- Subjects
- Hydrogen Bonding, Models, Molecular, Molecular Structure, Spectroscopy, Fourier Transform Infrared, ras GTPase-Activating Proteins metabolism, Guanosine Diphosphate chemistry, Phosphates chemistry, ras GTPase-Activating Proteins chemistry
- Abstract
Essential biochemical processes such as signal transduction, energy conversion, or substrate conversion depend on transient ligand binding. Thus, identifying the detailed structure and transient positioning of small ligands, and their stabilization by the surrounding protein, is of great importance. In this study, by decoding information from Fourier transform infrared (FTIR) spectra with biomolecular simulation methods, we identify the precise position and hydrogen network of a small compound, the guanosine diphosphate (GDP)-H(2)PO(4)(-) intermediate, in the surrounding protein-protein complex of Ras and its GTPase-activating protein, a central molecular switch in cellular signal transduction. We validate the simulated structure by comparing the calculated fingerprint vibrational modes of H(2)PO(4)(-) with those obtained from FTIR experiments. The new structural information, below the resolution of X-ray structural analysis, gives detailed insight into the catalytic mechanism.
- Published
- 2012
- Full Text
- View/download PDF
46. N-Ras forms dimers at POPC membranes.
- Author
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Güldenhaupt J, Rudack T, Bachler P, Mann D, Triola G, Waldmann H, Kötting C, and Gerwert K
- Subjects
- Fluorescence Resonance Energy Transfer, Lipid Bilayers metabolism, Molecular Dynamics Simulation, Mutation, Protein Structure, Quaternary, Spectroscopy, Fourier Transform Infrared, Surface Properties, ras Proteins genetics, ras Proteins metabolism, Phosphatidylcholines metabolism, Protein Multimerization, ras Proteins chemistry
- Abstract
Ras is a central regulator of cellular signaling pathways. It is mutated in 20-30% of human tumors. To perform its function, Ras has to be bound to a membrane by a posttranslationally attached lipid anchor. Surprisingly, we identified here dimerization of membrane anchored Ras by combining attenuated total reflectance Fourier transform infrared spectroscopy, biomolecular simulations, and Förster resonance energy transfer experiments. By analyzing x-ray structural models and molecular-dynamics simulations, we propose a dimerization interface between α-helices 4 and 5 and the loop between β2 and β3. This seems to explain why the residues D47, E49, R135, R161, and R164 of this interface are influencing Ras signaling in cellular physiological experiments, although they are not positioned in the catalytic site. Dimerization could catalyze nanoclustering, which is well accepted for membrane-bound Ras. The interface could provide a new target for a seemingly novel type of small molecule interfering with signal transduction in oncogenic Ras mutants., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
47. Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations.
- Author
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Rudack T, Xia F, Schlitter J, Kötting C, and Gerwert K
- Subjects
- Catalysis, Computer Simulation, Humans, Hydrolysis, Ligands, Magnesium chemistry, Molecular Conformation, Molecular Dynamics Simulation, Oxygen chemistry, Phosphates chemistry, Protein Conformation, Protons, Spectrophotometry, Infrared methods, Thermodynamics, X-Rays, ras Proteins metabolism, GTPase-Activating Proteins genetics, GTPase-Activating Proteins metabolism, Guanosine Triphosphate chemistry, Spectroscopy, Fourier Transform Infrared methods
- Abstract
Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and P(i) by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 10(5) compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 10(5) compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with time-resolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ- relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α- relative to the β- and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.
- Published
- 2012
- Full Text
- View/download PDF
48. Surface-attached polyhistidine-tag proteins characterized by FTIR difference spectroscopy.
- Author
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Pinkerneil P, Güldenhaupt J, Gerwert K, and Kötting C
- Subjects
- Germanium chemistry, Immobilized Proteins chemistry, Lipid Bilayers chemistry, Lipids chemistry, Nitrilotriacetic Acid chemistry, Proteins metabolism, Spectroscopy, Fourier Transform Infrared, Surface Properties, Histidine chemistry, Proteins chemistry
- Abstract
A universal label-free method for the spectroscopic investigation of polyhistidine-tagged proteins is presented. A solid supported lipid bilayer (SSLB, picture) containing nitrilotriacetic-acid-modified lipids is attached on top of a germanium attenuated total reflection crystal by hydrophilic interactions. Any His tag-modified protein can be immobilized and investigated by FTIR spectroscopy., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
- Full Text
- View/download PDF
49. The role of magnesium for geometry and charge in GTP hydrolysis, revealed by quantum mechanics/molecular mechanics simulations.
- Author
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Rudack T, Xia F, Schlitter J, Kötting C, and Gerwert K
- Subjects
- Biocatalysis, GTPase-Activating Proteins chemistry, Guanosine Diphosphate chemistry, Hydrolysis, Tyrosine chemistry, Vibration, Water chemistry, ras Proteins chemistry, Guanosine Triphosphate chemistry, Magnesium chemistry, Molecular Conformation, Molecular Dynamics Simulation, Quantum Theory
- Abstract
The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and P(i) in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and P(i) release back to the diphosphate. Furthermore, the Ras·Mg(2+) complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
50. Time-resolved Fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding Cassette transporter MsbA: ATP hydrolysis is the rate-limiting step in the catalytic cycle.
- Author
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Syberg F, Suveyzdis Y, Kötting C, Gerwert K, and Hofmann E
- Subjects
- ATP Binding Cassette Transporter, Subfamily B metabolism, ATP-Binding Cassette Transporters genetics, Bacterial Proteins genetics, Binding Sites genetics, Biological Transport, Catalysis, Escherichia coli genetics, Escherichia coli metabolism, Humans, Hydrolysis, Kinetics, Oxygen Isotopes, Phosphates metabolism, Time Factors, ATP-Binding Cassette Transporters metabolism, Adenosine Triphosphate metabolism, Bacterial Proteins metabolism, Spectroscopy, Fourier Transform Infrared methods
- Abstract
MsbA is an essential Escherichia coli ATP-binding cassette (ABC) transporter involved in the flipping of lipid A across the cytoplasmic membrane. It is a close homologue of human P-glycoprotein involved in multidrug resistance, and it similarly accepts a variety of small hydrophobic xenobiotics as transport substrates. X-ray structures of three full-length ABC multidrug exporters (including MsbA) have been published recently and reveal large conformational changes during the transport cycle. However, how ATP hydrolysis couples to these conformational changes and finally the transport is still an open question. We employed time-resolved FTIR spectroscopy, a powerful method to elucidate molecular reaction mechanisms of soluble and membrane proteins, to address this question with high spatiotemporal resolution. Here, we monitored the hydrolysis reaction in the nucleotide-binding domain of MsbA at the atomic level. The isolated MsbA nucleotide-binding domain hydrolyzed ATP with V(max) = 45 nmol mg(-1) min(-1), similar to the full-length transporter. A Hill coefficient of 1.49 demonstrates positive cooperativity between the two catalytic sites formed upon dimerization. Global fit analysis of time-resolved FTIR data revealed two apparent rate constants of ~1 and 0.01 s(-1), which were assigned to formation of the catalytic site and hydrolysis, respectively. Using isotopically labeled ATP, we identified specific marker bands for protein-bound ATP (1245 cm(-1)), ADP (1101 and 1205 cm(-1)), and free phosphate (1078 cm(-1)). Cleavage of the β-phosphate-γ-phosphate bond was found to be the rate-limiting step; no protein-bound phosphate intermediate was resolved.
- Published
- 2012
- Full Text
- View/download PDF
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