FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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References
Light-Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-trans and 13-cis Retinal Isomers
Bruun(*), S., Stöppler, D., Keidel(*), A., Kuhlmann(*), U., Luck(*), M., Diehl, A., Geiger, M. A., Woodmansee(*), D., Trauner(*), D., Hegemann(*), P., Oschkinat, H., Hildebrandt(*), P.; Stehfes(*)t, K.
Biochemistry, 54:5389-5400
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named "light adaptation". The main objective of this spectroscopic study was to elucidate the molecular processes associated with light-dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all-trans configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all-trans,15-anti and 13-cis,15-syn configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13) horizontal lineC(14) and C(15) horizontal lineN bonds. Both isomers of this DAapp state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13) horizontal lineC(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DAapp) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-trans isomer than for the 13-cis isomer, possibly because of different chromophore-water interactions. Our data demonstrating two complementary photocycles of the DAapp isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light-dark adaptation, as suggested previously by electrical measurements.

Theoretical aspects of Magic Angle Spinning - Dynamic Nuclear Polarization
Mentink-Vigier(*), F., Akbey, Ü., Oschkinat, H., Vega(*), S.; Feintuch(*), A.
J Magn Reson, 258:102-120
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Magic Angle Spinning (MAS) combined with Dynamic Nuclear Polarization (DNP) has been proven in recent years to be a very powerful method for increasing solid-state NMR signals. Since the advent of biradicals such as TOTAPOL to increase the nuclear polarization new classes of radicals, with larger molecular weight and/or different spin properties have been developed. These have led to unprecedented signal gain, with varying results for different experimental parameters, in particular the microwave irradiation strength, the static field, and the spinning frequency. Recently it has been demonstrated that sample spinning imposes DNP enhancement processes that differ from the active DNP mechanism in static samples as upon sample spinning the DNP enhancements are the results of energy level anticrossings occurring periodically during each rotor cycle. In this work we present experimental results with regards to the MAS frequency dependence of the DNP enhancement profiles of four nitroxide-based radicals at two different sets of temperature, 110 and 160K. In fact, different magnitudes of reduction in enhancement are observed with increasing spinning frequency. Our simulation code for calculating MAS-DNP powder enhancements of small model spin systems has been improved to extend our studies of the influence of the interaction and relaxation parameters on powder enhancements. To achieve a better understanding we simulated the spin dynamics of a single three-spin system ea-eb-n during its steady state rotor periods and used the Landau-Zener formula to characterize the influence of the different anti-crossings on the polarizations of the system and their necessary action for reaching steady state conditions together with spin relaxation processes. Based on these model calculations we demonstrate that the maximum steady state nuclear polarization cannot become larger than the maximum polarization difference between the two electrons during the steady state rotor cycle. This study also shows the complexity of the MAS-DNP process and therefore the necessity to rely on numerical simulations for understanding parametric dependencies of the enhancements. Finally an extension of the spin system up to five spins allowed us to probe the first steps of the transfer of polarization from the nuclei coupled to the electrons to further away nuclei, demonstrating a decrease in the spin-diffusion barrier under MAS conditions.

Alterations in creatine metabolism observed in experimental autoimmune myocarditis using ex vivo proton magic angle spinning MRS
Muench(*), F., Retel, J., Jeuthe(*), S., (*)D, O. h.-I., van Rossum, B., Wassilew(*), K., Schmerler(*), P., Kuehne(*), T., Berger(*), F., Oschkinat, H.; Messroghli(*), D. R.
Nmr Biomed, 28:1625-1633
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Experimental autoimmune myocarditis (EAM) in rodents is an accepted model of myocarditis and dilated cardiomyopathy (DCM). Altered metabolism is thought to play an important role in the pathogenesis of DCM and heart failure (HF). Study of the metabolism may provide new diagnostic information and insights into the mechanisms of myocarditis and HF. Proton MRS ((1)H-MRS) has not yet been used to study the changes occurring in myocarditis and subsequent HF. We aimed to explore the changes in creatine metabolism using this model and compare them with the findings in healthy animals. Myocardial function of male young Lewis rats with EAM was quantified by performing left ventricular ejection fraction (LVEF) analysis in short-axis cine images throughout the whole heart. Inflammatory cellular infiltrate was assessed by immunohistochemistry. Myocardial tissue was analyzed using ex vivo proton magic angle spinning MRS ((1)H-MAS-MRS). Myocarditis was confirmed histologically by the presence of an inflammatory cellular infiltrate and CD68 positive staining. A significant increase in the metabolic ratio of Tau/tCr (taurine/total creatine) obtained by (1)H-MAS-MRS was observed in myocarditis compared with healthy controls (21 d acute EAM, 4.38 (+/-0.23); 21 d control, 2.84 (+/-0.08); 35 d chronic EAM, 4.47 (+/-0.83); 35 d control, 2.59 (+/-0.38); P < 0.001). LVEF was reduced in diseased animals (EAM, 55.2% (+/-11.3%); control, 72.6% (+/-3.8%); P < 0.01) and correlated with Tau/tCr ratio (R = 0.937, P < 0.001). Metabolic alterations occur acutely with the development of myocarditis. Myocardial Tau/tCr ratio as detected by (1)H-MRS correlates with LVEF and is able to differentiate between healthy myocardium and myocardium from rats with EAM.

Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning
Nieuwkoop, A. J., Franks, W. T., Rehbein, K., Diehl, A., Akbey, Ü., Engelke(*), F., Emsley(*), L., Pintacuda(*), G.; Oschkinat, H.
J Biomol NMR, 61:161-171
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The use of small rotors capable of very fast magic-angle spinning (MAS) in conjunction with proton dilution by perdeuteration and partial reprotonation at exchangeable sites has enabled the acquisition of resolved, proton detected, solid-state NMR spectra on samples of biological macromolecules. The ability to detect the high-gamma protons, instead of carbons or nitrogens, increases sensitivity. In order to achieve sufficient resolution of the amide proton signals, rotors must be spun at the maximum rate possible given their size and the proton back-exchange percentage tuned. Here we investigate the optimal proton back-exchange ratio for triply labeled SH3 at 40 kHz MAS. We find that spectra acquired on 60 % back-exchanged samples in 1.9 mm rotors have similar resolution at 40 kHz MAS as spectra of 100 % back-exchanged samples in 1.3 mm rotors spinning at 60 kHz MAS, and for (H)NH 2D and (H)CNH 3D spectra, show 10-20 % higher sensitivity. For 100 % back-exchanged samples, the sensitivity in 1.9 mm rotors is superior by a factor of 1.9 in (H)NH and 1.8 in (H)CNH spectra but at lower resolution. For (H)C(C)NH experiments with a carbon-carbon mixing period, this sensitivity gain is lost due to shorter relaxation times and less efficient transfer steps. We present a detailed study on the sensitivity of these types of experiments for both types of rotors, which should enable experimentalists to make an informed decision about which type of rotor is best for specific applications.

A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions
Opitz, R., Müller, M., Reuter, C., Barone, M., Soicke(*), A., Roske(*), Y., Piotukh, K., Huy(*), P., Beerbaum, M., Wiesner, B., Beyermann, M., Schmieder, P., Freund(*), C., Volkmer, R., Oschkinat, H., Schmalz(*), H. G.; Kühne, R.
Proc Natl Acad Sci U S A, 112:5011-5016
(2015)

Tags: Computational Chemistry and Protein Design (Kühne), NMR-Supported Structural Biology (Oschkinat), Peptide Chemistry (Hackenberger/ Volkmer), Solution NMR (Schmieder), Peptide Chemistry (Beyermann), Cellular Imaging (Wiesner)

Abstract: Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

Design and Stereoselective Synthesis of ProM-2: A Spirocyclic Diproline Mimetic with Polyproline Type II (PPII) Helix Conformation
Reuter(*), C., Opitz, R., Soicke(*), A., Dohmen(*), S., Barone, M., Chiha(*), S., Klein(*), M. T., Neudörfl(*), J. M., Kühne, R.; Schmalz(*), H. G.
Chemistry, 21:8464-8470
(2015)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: With the aim of developing polyproline type II helix (PPII) secondary-structure mimetics for the modulation of prolin-rich-mediated protein-protein interactions, the novel diproline mimetic ProM-2 was designed by bridging the two pyrrolidine rings of a diproline (Pro-Pro) unit through a Z-vinylidene moiety. This scaffold, which closely resembles a section of a PPII helix, was then stereoselectively synthesized by exploiting a ruthenium-catalyzed ring-closing metathesis (RCM) as a late key step. The required vinylproline building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester, were prepared on a gram scale as pure stereoisomers. The difficult peptide coupling of the sterically demanding building blocks was achieved in good yield and without epimerization by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIPEA). The RCM proceeded smoothly in the presence of the Grubbs II catalyst. Stereostructural assignments for several intermediates were secured by X-ray crystallography. As a proof of concept, it was shown that certain peptides containing ProM-2 exhibited improved (canonical) binding towards the Ena/VASP homology 1 (EVH1) domain as a relevant protein interaction target.

Simian hemorrhagic fever virus cell entry is dependent on CD163 and uses a clathrin-mediated endocytosis-like pathway
Cai(*), Y., Postnikova(*), E. N., Bernbaum(*), J. G., Yu(*), S. Q., Mazur(*), S., Deiuliis(*), N. M., Radoshitzky(*), S. R., Lackemeyer(*), M. G., McCluskey(*), A., Robinson(*), P. J., Haucke, V., Wahl-Jensen(*), V., Bailey(*), A. L., Lauck(*), M., Friedrich(*), T. C., O'Connor(*), D. H., Goldberg(*), T. L., Jahrling(*), P. B.; Kuhn(*), J. H.
J Virol, 89:844-856
(2015)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: UNLABELLED: Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-beta-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, alpha-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component. IMPORTANCE: Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa virus. However, little is known about SHFV's ecology and molecular biology and the mechanism by which it causes disease. The results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low-pH-dependent, actin-independent endocytosis, likely with the help of a cellular surface protein.

Thermodynamics of protein destabilization in live cells
Danielsson(*), J., Mu(*), X., Lang(*), L., Wang(*), H., Binolfi, A., Theillet, F. X., Bekei, B., Logan(*), D. T., Selenko, P., Wennerstrom(*), H.; Oliveberg(*), M.
Proc Natl Acad Sci U S A, 112:12402-12407
(2015)

Tags: In-Cell NMR (Selenko)

Abstract: Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a beta-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 degrees C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Self-assembly Properties
Herrada(*), I., Samson(*), C., Velours(*), C., Renault(*), L., Ostlund(*), C., Chervy(*), P., Puchkov, D., Worman(*), H. J., Buendia(*), B.; Zinn-Justin(*), S.
ACS Chem Biol, 10:2733-2742
(2015)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: More than 100 genetic mutations causing X-linked Emery-Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding emerin variants' loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of beta-structures. Analysis of the assembly properties of five EmN variants reveals that del95-99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of emerin-emerin and emerin-lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to emerin aggregation and mislocalization in cells. Our results suggest that emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.

Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics
Serwa(*), R. A., Abaitua(*), F., Krause, E., Tate(*), E. W.; O'Hare(*), P.
Chem Biol, 22:1008-1017
(2015)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a generalized reduction in myristoylation of host proteins, whereas palmitoylation of host proteins, including regulators of interferon and tetraspanin family proteins, was selectively repressed. Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions.

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