FMP Publications

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

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References
Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins
del Amo, J. M. L., Agarwal, V., Sarkar(*), R., Porter(*), J., Asami(*), S., Rubbelke(*), M., Fink, U., Xue(*), Y., Lange(*), O. F.; Reif, B.
J. Biomol. NMR, 59:241-249
(2014)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Relaxation parameters such as longitudinal relaxation are susceptible to artifacts such as spin diffusion, and can be affected by paramagnetic impurities as e.g. oxygen, which make a quantitative interpretation difficult. We present here the site-specific measurement of [H-1]C-13 and [H-1]N-15 heteronuclear rates in an immobilized protein. For methyls, a strong effect is expected due to the three-fold rotation of the methyl group. Quantification of the [H-1]C-13 heteronuclear NOE in combination with C-13-R (1) can yield a more accurate analysis of side chain motional parameters. The observation of significant [H-1]N-15 heteronuclear NOEs for certain backbone amides, as well as for specific asparagine/glutamine sidechain amides is consistent with MD simulations. The measurement of site-specific heteronuclear NOEs is enabled by the use of highly deuterated microcrystalline protein samples in which spin diffusion is reduced in comparison to protonated samples.

Chemoselective Wittig and Michael Ligations of Unprotected Peptidyl Phosphoranes in Water Furnish Potent Inhibitors of Caspase-3
Holland-Nell, K., Fernandez-Bachiller, M. I., Ahsanullah, R.J.; Rademann, J.
Org Lett, 16:4428-4431
(2014)

Tags: Medicinal Chemistry (Rademann)

Abstract: Unprotected peptidyl phosphoranes 1 with sequence Ac-L-aspartyl-L-glutamyl-L-valinyl-L-aspartyl are released from polymer support and react with aliphatic and aromatic aldehydes in aqueous medium in a Wittig ligation. Obtained vinyl ketones 6-12 are potent inhibitors of caspase-3. Vinyl ketone 6, derived from formaldehyde, undergoes Michael ligations with thiol nucleophiles furnishing products 14-16, also in aqueous medium. The demonstrated ligation reactions enable the modification of complex functionalized peptides in water providing bioactive protein ligands without side-chain protection.

Low-power polarization transfer between deuterons and spin-1/2 nuclei using adiabatic (CP)-C-RESPIRATION in solid-state NMR
Jain(*), S. K., Nielsen(*), A. B., Hiller, M., Handel, L., Ernst(*), M., Oschkinat, H., Akbey, Ü.; Nielsen(*), N. C.
Physical Chemistry Chemical Physics, 16:2827-2830
(2014)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Establishing high-resolution structures of biological macromolecules in heterogeneous environments by MAS solid-state NMR is an important challenge where development of advanced experimental procedures is in high demand. Promising new methods take advantage of samples with extensive H-2, C-13, and N-15 isotope labelling, effectively diluting 1H spins. In many cases, a sufficient amount of H-1 at exchangeable sites cannot be re-established during the purification procedure, hence it is necessary to exploit also the potential of H-2 as a starting point in pulse sequences, capitalizing on its short T-1 as compared to C-13, and to detect carbon or proton spins as appropriate. Here we present a new method that enables the required high-efficiency H-2, C-13, and N-15 polarization transfer to be accomplished under the limited H-2 rf power conditions using current H-1, H-2, C-13 and N-15 quadruple-resonance MAS NMR instrumentation.

Quadruple-resonance magic-angle spinning NMR spectroscopy of deuterated solid proteins
Akbey, Ü., Nieuwkoop, A. J., Wegner, S., Voreck, A., Kunert, B., Bandara, P., Engelke, F., Nielsen, N. C.; Oschkinat, H.
Angew Chem Int Ed Engl, 53:2438-2442
(2014)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: (1)H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using (2)H excitation instead of (1)H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, "quadruple-resonance NMR spectroscopy", is presented which relies on an efficient (2)H-excitation and (2)H-(13)C cross-polarization (CP) step, combined with (1)H detection. We show that by using (2)H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.

Flexible, polymer-supported synthesis of sphingosine derivatives provides ceramides with enhanced biological activity
El-Dahshan, A., Al-Gharabli(*), S. I., Radetzki, S., Al-Tel(*), T. H., Kumar(*), P.; Rademann, J.
Bioorg Med Chem, 22:5506-5512
(2014)

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries)

Abstract: A polymer-supported route for the synthesis of sphingosine derivatives is presented based on the C-acylation of polymeric phosphoranylidene acetates with an Fmoc-protected amino acid. The approach enables the flexible variation of the sphingosine tail through a deprotection-decarboxylation sequence followed by E-selective Wittig olefination cleavage. d-Erythro-sphingosine analogs have been synthesized by diastereoselective reduction of the keto group employing LiAlH(O-tBu)3 as reducing agent. The effect of ceramides and keto-ceramides on the proliferation of three cancer cell lines HEP G-2, PC-12 and HL-60 was investigated and a ceramide containing an aromatic sphingosine tail was identified as being most active.

Semisynthesis and optimization of G protein-coupled receptor mimics
Abel, S., Geltinger, B., Heinrich, N., Michl, D., Klose, A., Beyermann, M.; Schwarzer(*), D.
J Pept Sci, 20:831-836
(2014)

Tags: Peptide Chemistry (Beyermann)

Abstract: We have recently developed a soluble mimic of the corticotropin-releasing factor receptor type 1 (CRF1), a membrane-spanning G protein-coupled receptor, which allowed investigations on receptor-ligand interactions. The CRF1 mimic consists of the receptor N-terminus and three synthetic extracellular loops (ECL1-3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost-efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop-mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs.

Interrelation between protein synthesis, proteostasis and life span
Arnsburg, K.; Kirstein-Miles, J.
Current genomics, 15:66-75
(2014)

Tags: Proteostasis in Aging and Disease (Kirstein)

Abstract: The production of newly synthesized proteins is a key process of protein homeostasis that initiates the biosynthetic flux of proteins and thereby determines the composition, stability and functionality of the proteome. Protein synthesis is highly regulated on multiple levels to adapt the proteome to environmental and physiological challenges such as aging and proteotoxic conditions. Imbalances of protein folding conditions are sensed by the cell that then trigger a cascade of signaling pathways aiming to restore the protein folding equilibrium. One regulatory node to rebalance proteostasis upon stress is the control of protein synthesis itself. Translation is reduced as an immediate response to perturbations of the protein folding equilibrium that can be observed in the cytosol as well as in the organelles such as the endoplasmatic reticulum and mitochondria. As reduction of protein synthesis is linked to life span increase, the signaling pathways regu-lating protein synthesis might be putative targets for treatments of age-related diseases. Eukaryotic cells have evolved a complex system for protein synthesis regulation and this review will summarize cellular strategies to regulate mRNA translation upon stress and its impact on longevity.

Rapid proton-detected NMR assignment for proteins with fast magic angle spinning
Barbet-Massin(*), E., Pell(*), A. J., Retel, J. S., Andreas(*), L. B., Jaudzems(*), K., Franks, W. T., Nieuwkoop, A. J., Hiller, M., Higman(*), V., Guerry(*), P., Bertarello(*), A., Knight(*), M. J., Felletti(*), M., Le Marchand(*), T., Kotelovica(*), S., Akopjana(*), I., Tars(*), K., Stoppini(*), M., Bellotti(*), V., Bolognesi(*), M., Ricagno(*), S., Chou(*), J. J., Griffin(*), R. G., Oschkinat, H., Lesage(*), A., Emsley(*), L., Herrmann(*), T.; Pintacuda(*), G.
J Am Chem Soc, 136:12489-12497
(2014)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (omegar/2pi >/= 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary (13)C/(15)N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

CLCN7 and TCIRG1 Mutations Differentially Affect Bone Matrix Mineralization in Osteopetrotic Individuals
Barvencik(*), F., Kurth(*), I., Koehne(*), T., Stauber, T., Zustin(*), J., Tsiakas, K., Ludwig, C. F., Beil(*), F. T., Pestka(*), J. M., Hahn(*), M., Santer(*), R., Supanchart(*), C., Kornak(*9, U., Del Fattore(*), A., Jentsch, T. J., Teti(*), A., Schulz(*), A., Schinke(*), T.; Amling(*), M.
J Bone Miner Res, 29:982-991
(2014)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity
Chakrabarti(*), A., Witsenburg(*), J. J., Sinzinger(*), M. D., Richter, M., Wallbrecher(*), R., Cluitmans(*), J. C., Verdurmen(*), W. P., Tanis(*), S., Adjobo-Hermans(*), M. J., Rademann, J.; Brock(*), R.
Biochim Biophys Acta, 1838:3097-3106
(2014)

Tags: Medicinal Chemistry (Rademann)

Abstract: Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10muM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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13125 Berlin, Germany
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info(at)fmp-berlin.de

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