FMP Publications

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

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References
Mode of action of cationic antimicrobial peptides defines the tethering position and the efficacy of biocidal surfaces
Bagheri, M., Beyermann, M.; Dathe, M.
Bioconjug Chem, 23:66-74
(2012)

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe), Peptide Chemistry (Beyermann)

Abstract: Covalent immobilization of cationic antimicrobial peptides (CAPs) at sufficient density and distance from the solid matrix has been suggested as a successful strategy for the generation of biocidal surfaces. To test the hypothesis that the mode of peptide action is decisive for the selection of an appropriate tethering position on solid surfaces, melittin (MEL), a channel-forming peptide, buforin 2 (BUF2), a peptide able to translocate bacterial membranes without permeabilization and targeting nucleic acids, and tritrpticin (TP), described to be membrane-lytic and to have intracellular targets, were C- and N-terminally immobilized on TentaGel S NH(2) resin beads as model surface. The peptide termini were modified with aminooxyacetic acid (AOA) and coupled via oxime-forming ligation. The comparison of the activities of the three peptides and their AOA-modified analogues with a KLAL model peptide which permeabilizes membranes by a so-called "carpet-like" mode provided the following results: The peptides in solution state were active against Bacillus subtilis and Escherichia coli at micromolar concentrations. MEL and TP but not BUF2-derived peptides permeabilized the inner and outer membrane of E. coli and enhanced the permeability of lipid bilayers at concentrations around their antimicrobial values (MICs). Immobilization reduced peptide activity to millimolar MICs. The activity reduction for KLAL was independent of the tethering position and comparably low, as reflected by a low ratio of MIC(tethered)/MIC(free). In contrary, the pore-forming MEL was much less active when immobilized at the N-terminus compared with the C-terminally tethered peptide. C- and N-terminal TP tethering caused an identical but much pronounced activity decrease. The tethered BUF2 peptides were inactive at the tested concentrations suggesting that the peptides could not reach the intracellular targets. In conclusion, membrane active peptides seem to be most suitable for the generation of antimicrobial surfaces, but knowledge about their mode of membrane insertion and positioning is required to identify optimal tethering positions. The relationship between the mechanism of action and position of immobilization is highly relevant for the establishment of a general approach to obtain efficient biocidal solid matrices loaded with CAPs.

Soluble peptidyl phosphoranes for metal-free, stereoselective ligations in organic and aqueous solution
Ahsanullah, R.J., Al-Gharabli, S. I.; Rademann, J.
Org Lett, 14:14-17
(2012)

Tags: Medicinal Chemistry (Rademann)

Abstract: Protocols for solid-phase syntheses of soluble peptidyl phosphoranes are presented. Various supported phosphoranylidene acetates were prepared on Rink amide or via alkylation of trialkyl- and triarylphosphines with bromoacetyl Wang ester. C-Acylation was conducted racemization-free with activated Fmoc-amino acids, followed by SPPS (solid-phase peptide synthesis). Acidic conditions released decarboxylated peptidyl phosphoranes into solution. The protocol allowed for the electronic variation of peptidyl phosphoranes which were investigated in ligation reactions with azides in organic and aqueous solvents.

High-Temperature Dynamic Nuclear Polarization Enhanced Magic-Angle-Spinning NMR
Akbey, Ü., Linden, A. H.; Oschkinat, H.
Appl Magn Reson, 43:81-90
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic nuclear polarization (DNP) transfers electron spin-polarization to nuclear spins in close proximity, increasing sensitivity by two-to-three orders of magnitude. This enables nuclear magnetic resonance (NMR) experiments on samples with low concentrations of analyte. The requirement of using cryogenic temperatures in DNP-enhanced solid-state NMR (ssNMR) experiments may impair the resolution and hence limit its broad application to biological systems. In this work, we introduce a "High-Temperature DNP" approach, which aims at increasing spectral resolution by performing experiments at temperatures of around 180 K instead of similar to 100 K. By utilizing the extraordinary enhancements obtained on deuterated proteins, still sufficiently large DNP enhancements of 11-18 are obtained for proton and carbon, respectively. We recorded high sensitivity 2D C-13-C-13 spectra in similar to 9 min with higher resolution than at 100 K, which has similar resolution to the one obtained at room temperature for some favorable residues.

Practical aspects of high-sensitivity multidimensional (1)(3)C MAS NMR spectroscopy of perdeuterated proteins
Akbey, Ü., Rossum, B. J.; Oschkinat, H.
J Magn Reson, 217:77-85
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The double nucleus enhanced recoupling (DONER) experiment employs simultaneous irradiation of protons and deuterons to promote spin diffusion processes in a perdeuterated protein. This results in 4-5 times higher sensitivity in 2D (13)C-(13)C correlation experiments as compared to PDSD [1]. Here, a quantitative comparison of PDSD, (1)H-DARR, (2)H-DARR, and (1)H+(2)H DONER has been performed to analyze the influence of spin diffusion on polarization transfer processes. Cross peak buildup curves were analyzed to obtain guidelines for choosing the best experimental parameters. The largest cross peak intensities were observed for the DONER experiments. The fastest build-up rate was observed in the (2)H-DARR experiment within a buildup range of approximately 18-45 ms, whereas values between 24 and 69 ms are observed for the DONER experiment. Furthermore, the effects of direct excitation and cross polarization (CP) are compared. A comparison between DONER and RFDR experiments reveal approximately 50% more intense cross peaks in the C(alpha)-CO and C(alpha)-C(alip) regions of the 2D (13)C-(13)C DONER spectrum applying proton CP ((1)H-(13)C). As a parameter determining the S/N in (13)C-(13)C correlation experiments, proton CP efficiency is investigated using deuterated samples with proton/deuterium ratios at 20%, 40%, and 100% H(2)O. Sufficiently strong (13)C CPMAS signal intensity is observed for such proteins even with very low proton concentration. The effect of proton and/or deuterium decoupling is analyzed at various MAS spinning frequencies. Deuterium decoupling was found most crucial for obtaining high resolution. Long range correlations are readily observed representing distances up to approximately 6 A by using DONER approach.

Assignment strategies for aliphatic protons in the solid-state in randomly protonated proteins
Asami, S.; Reif, B.
J. Biomol. NMR, 52:31-39
(2012)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Biological solid-state nuclear magnetic resonance spectroscopy developed rapidly in the past two decades and emerged as an important tool for structural biology. Resonance assignment is an essential prerequisite for structure determination and the characterization of motional properties of a molecule. Experiments, which rely on carbon or nitrogen detection, suffer, however, from low sensitivity. Recently, we introduced the RAP (Reduced Adjoining Protonation) labeling scheme, which allows to detect backbone and sidechain protons with high sensitivity and resolution. We present here a H-1-detected 3D (H)CCH experiment for assignment of backbone and sidechain proton resonances. Resolution is significantly improved by employing simultaneous (CO)-C-13 and C-13 beta J-decoupling during evolution of the C-13 alpha chemical shift. In total, similar to 90% of the H-1 alpha-C-13 alpha backbone resonances of chicken alpha-spectrin SH3 could be assigned.

Optimal degree of protonation for (1)H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency
Asami, S., Szekely(*), K., Schanda(*), P., Meier(*), B. H.; Reif, B.
J Biomol NMR, 54:155-168
(2012)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The (1)H dipolar network, which is the major obstacle for applying proton detection in the solid-state, can be reduced by deuteration, employing the RAP (Reduced Adjoining Protonation) labeling scheme, which yields random protonation at non-exchangeable sites. We present here a systematic study on the optimal degree of random sidechain protonation in RAP samples as a function of the MAS (magic angle spinning) frequency. In particular, we compare (1)H sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken alpha-spectrin, for samples, prepared with 5-25 % H(2)O in the E. coli growth medium, in the MAS frequency range of 20-60 kHz. At an external field of 19.96 T (850 MHz), we find that using a proton concentration between 15 and 25 % in the M9 medium yields the best compromise in terms of sensitivity and resolution, with an achievable average (1)H linewidth on the order of 40-50 Hz. Comparing sensitivities at a MAS frequency of 60 versus 20 kHz, a gain in sensitivity by a factor of 4-4.5 is observed in INEPT-based (1)H detected 1D (1)H,(13)C correlation experiments. In total, we find that spectra recorded with a 1.3 mm rotor at 60 kHz have almost the same sensitivity as spectra recorded with a fully packed 3.2 mm rotor at 20 kHz, even though ~20x less material is employed. The improved sensitivity is attributed to (1)H line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.

Optimal degree of protonation for H-1 detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency
Asami, S., Szekely(*), K., Schanda(*), P., Meier(*), B. H.; Reif, B.
J. Biomol. NMR, 54:155-168
(2012)

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The H-1 dipolar network, which is the major obstacle for applying proton detection in the solid-state, can be reduced by deuteration, employing the RAP (Reduced Adjoining Protonation) labeling scheme, which yields random protonation at non-exchangeable sites. We present here a systematic study on the optimal degree of random sidechain protonation in RAP samples as a function of the MAS (magic angle spinning) frequency. In particular, we compare H-1 sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken alpha-spectrin, for samples, prepared with 5-25 % H2O in the E. coli growth medium, in the MAS frequency range of 20-60 kHz. At an external field of 19.96 T (850 MHz), we find that using a proton concentration between 15 and 25 % in the M9 medium yields the best compromise in terms of sensitivity and resolution, with an achievable average H-1 linewidth on the order of 40-50 Hz. Comparing sensitivities at a MAS frequency of 60 versus 20 kHz, a gain in sensitivity by a factor of 4-4.5 is observed in INEPT-based H-1 detected 1D H-1,C-13 correlation experiments. In total, we find that spectra recorded with a 1.3 mm rotor at 60 kHz have almost the same sensitivity as spectra recorded with a fully packed 3.2 mm rotor at 20 kHz, even though similar to 20x less material is employed. The improved sensitivity is attributed to H-1 line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.

Mode of action of cationic antimicrobial peptides defines the tethering position and the efficacy of biocidal surfaces
Bagheri, M., Beyermann, M.; Dathe, M.
Bioconjug Chem, 23:66-74
(2012)

Tags: Peptide Chemistry (Beyermann),Peptide-Lipide-Interaction (Dathe)

Abstract: Covalent immobilization of cationic antimicrobial peptides (CAPs) at sufficient density and distance from the solid matrix has been suggested as a successful strategy for the generation of biocidal surfaces. To test the hypothesis that the mode of peptide action is decisive for the selection of an appropriate tethering position on solid surfaces, melittin (MEL), a channel-forming peptide, buforin 2 (BUF2), a peptide able to translocate bacterial membranes without permeabilization and targeting nucleic acids, and tritrpticin (TP), described to be membrane-lytic and to have intracellular targets, were C- and N-terminally immobilized on TentaGel S NH(2) resin beads as model surface. The peptide termini were modified with aminooxyacetic acid (AOA) and coupled via oxime-forming ligation. The comparison of the activities of the three peptides and their AOA-modified analogues with a KLAL model peptide which permeabilizes membranes by a so-called "carpet-like" mode provided the following results: The peptides in solution state were active against Bacillus subtilis and Escherichia coli at micromolar concentrations. MEL and TP but not BUF2-derived peptides permeabilized the inner and outer membrane of E. coli and enhanced the permeability of lipid bilayers at concentrations around their antimicrobial values (MICs). Immobilization reduced peptide activity to millimolar MICs. The activity reduction for KLAL was independent of the tethering position and comparably low, as reflected by a low ratio of MIC(tethered)/MIC(free). In contrary, the pore-forming MEL was much less active when immobilized at the N-terminus compared with the C-terminally tethered peptide. C- and N-terminal TP tethering caused an identical but much pronounced activity decrease. The tethered BUF2 peptides were inactive at the tested concentrations suggesting that the peptides could not reach the intracellular targets. In conclusion, membrane active peptides seem to be most suitable for the generation of antimicrobial surfaces, but knowledge about their mode of membrane insertion and positioning is required to identify optimal tethering positions. The relationship between the mechanism of action and position of immobilization is highly relevant for the establishment of a general approach to obtain efficient biocidal solid matrices loaded with CAPs.

Molecular Evolution of a Peptide GPCR Ligand Driven by Artificial Neural Networks
Bandholtz(*), S., Wichard, J., Kühne, R.; Grotzinger(*), C.
Plos One, 7
(2012)

Tags: Computational Chemistry and Protein Design (Kühne)

Abstract: Peptide ligands of G protein-coupled receptors constitute valuable natural lead structures for the development of highly selective drugs and high-affinity tools to probe ligand-receptor interaction. Currently, pharmacological and metabolic modification of natural peptides involves either an iterative trial-and-error process based on structure-activity relationships or screening of peptide libraries that contain many structural variants of the native molecule. Here, we present a novel neural network architecture for the improvement of metabolic stability without loss of bioactivity. In this approach the peptide sequence determines the topology of the neural network and each cell corresponds one-to-one to a single amino acid of the peptide chain. Using a training set, the learning algorithm calculated weights for each cell. The resulting network calculated the fitness function in a genetic algorithm to explore the virtual space of all possible peptides. The network training was based on gradient descent techniques which rely on the efficient calculation of the gradient by back-propagation. After three consecutive cycles of sequence design by the neural network, peptide synthesis and bioassay this new approach yielded a ligand with 70fold higher metabolic stability compared to the wild type peptide without loss of the subnanomolar activity in the biological assay. Combining specialized neural networks with an exploration of the combinatorial amino acid sequence space by genetic algorithms represents a novel rational strategy for peptide design and optimization.

ARIA for solution and solid-state NMR
Bardiaux, B., Malliavin(*), T.; Nilges(*), M.
Methods Mol Biol, 831:453-483
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: In solution or solid-state, determining the three-dimensional structure of biomolecules by Nuclear -Magnetic Resonance (NMR) normally requires the collection of distance information. The interpretation of the spectra containing this distance information is a critical step in an NMR structure determination. In this chapter, we present the Ambiguous Restraints for Iterative Assignment (ARIA) program for automated cross-peak assignment and determination of macromolecular structure from solution and solid-state NMR experiments. While the program was initially designed for the assignment of nuclear Overhauser effect (NOE) resonances, it has been extended to the interpretation of magic-angle spinning (MAS) solid-state NMR data. This chapter first details the concepts and procedures carried out by the program. Then, we describe both the general strategy for structure determination with ARIA 2.3 and practical aspects of the technique. ARIA 2.3 includes all recent developments. such as an extended integration of the Collaborative Computing Project for the NMR community (CCPN), the incorporation of the log-harmonic distance restraint potential and an automated treatment of symmetric oligomers.

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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
+4930 94793 - 100 
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info(at)fmp-berlin.de

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