Publications by authors named "Anne S Ulrich"

192 Publications

Diarylethene-Based Photoswitchable Inhibitors of Serine Proteases.

Angew Chem Int Ed Engl 2021 Jul 16. Epub 2021 Jul 16.

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology (KIT), POB 3640, 76021, Karlsruhe, Germany.

A bicyclic peptide scaffold was chemically adapted to generate diarylethene-based photoswitchable inhibitors of serine protease Bos taurus trypsin 1 (T1). Starting from a prototype molecule-sunflower trypsin inhibitor-1 (SFTI-1)-we obtained light-controllable inhibitors of T1 with K in the low nanomolar range, whose activity could be modulated over 20-fold by irradiation. The inhibitory potency as well as resistance to proteolytic degradation were systematically studied on a series of 17 SFTI-1 analogues. The hydrogen bond network that stabilizes the structure of inhibitors and possibly the enzyme-inhibitor binding dynamics were affected by isomerization of the photoswitch. The feasibility of manipulating enzyme activity in time and space was demonstrated by controlled digestion of gelatin-based hydrogel and an antimicrobial peptide BP100-RW. Finally, our design principles of diarylethene photoswitches are shown to apply also for the development of other serine protease inhibitors.
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http://dx.doi.org/10.1002/anie.202108847DOI Listing
July 2021

Behavior of the Antibacterial Peptide Cyclo[RRRWFW], Explored Using a 3-Hydroxychromone-Derived Fluorescent Amino Acid.

Front Chem 2021 28;9:688446. Epub 2021 Jun 28.

Taras Shevchenko National University of Kyiv, Kyiv, Ukraine.

Labeling biomolecules with fluorescent labels is an established tool for structural, biochemical, and biophysical studies; however, it remains underused for small peptides. In this work, an amino acid bearing a 3-hydroxychromone fluorophore, 2-amino-3-(2-(furan-2-yl)-3-hydroxy-4-oxo-4H-chromen-6-yl)propanoic acid (FHC), was incorporated in a known hexameric antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues. Circular dichroism spectropolarimetry and antibacterial activity measurements demonstrated that the FHC residue perturbs the peptide structure depending on labeling position but does not modify the activity of cWFW significantly. FHC thus can be considered an adequate label for studies of the parent peptide. Several analytical and imaging techniques were used to establish the activity of the obtained labeled cWFW analogues toward animal cells and to study the behavior of the peptides in a multicellular organism. The 3-hydroxychromone fluorophore can undergo excited-state intramolecular proton transfer (ESIPT), resulting in double-band emission from its two tautomeric forms. This feature allowed us to get insights into conformational equilibria of the labeled peptides, localize the cWFW analogues in human cells (HeLa and HEK293) and zebrafish embryos, and assess the polarity of the local environment around the label by confocal fluorescence microscopy. We found that the labeled peptides efficiently penetrated cancerous cells and localized mainly in lipid-containing and/or other nonpolar subcellular compartments. In the zebrafish embryo, the peptides remained in the bloodstream upon injection into the cardinal vein, presumably adhering to lipoproteins and/or microvesicles. They did not diffuse into any tissue to a significant extent during the first 3 h after administration. This study demonstrated the utility of fluorescent labeling by double-emission labels to evaluate biologically active peptides as potential drug candidates .
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http://dx.doi.org/10.3389/fchem.2021.688446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8273159PMC
June 2021

Order and disorder-An integrative structure of the full-length human growth hormone receptor.

Sci Adv 2021 Jun 30;7(27). Epub 2021 Jun 30.

Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaløes vej 5, 2200 Copenhagen N, Denmark.

Because of its small size (70 kilodalton) and large content of structural disorder (>50%), the human growth hormone receptor (hGHR) falls between the cracks of conventional high-resolution structural biology methods. Here, we study the structure of the full-length hGHR in nanodiscs with small-angle x-ray scattering (SAXS) as the foundation. We develop an approach that combines SAXS, x-ray diffraction, and NMR spectroscopy data obtained on individual domains and integrate these through molecular dynamics simulations to interpret SAXS data on the full-length hGHR in nanodiscs. The hGHR domains reorient freely, resulting in a broad structural ensemble, emphasizing the need to take an ensemble view on signaling of relevance to disease states. The structure provides the first experimental model of any full-length cytokine receptor in a lipid membrane and exemplifies how integrating experimental data from several techniques computationally may access structures of membrane proteins with long, disordered regions, a widespread phenomenon in biology.
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http://dx.doi.org/10.1126/sciadv.abh3805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245047PMC
June 2021

Chiral Resolution of Spin-Crossover Active Iron(II) [2x2] Grid Complexes.

Chemistry 2021 Jun 24. Epub 2021 Jun 24.

Institute of Nanotechnology (INT), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.

Chiral magnetic materials are proposed for applications in second-order non-linear optics, magneto-chiral dichroism, among others. Recently, we have reported a set of tetra-nuclear Fe(II) grid complex conformers with general formula C/S-[Fe L ] (L: 2,6-bis(6-(pyrazol-1-yl)pyridin-2-yl)-1,5-dihydrobenzo[1,2-d : 4,5-d']diimidazole). In the grid complexes, isomerism emerges from tautomerism and conformational isomerism of the ligand L, and the S-type grid complex is chiral, which originates from different non-centrosymmetric spatial organization of the trans type ligand around the Fe(II) center. However, the selective preparation of an enantiomerically pure grid complex in a controlled manner is difficult due to spontaneous self-assembly. To achieve the pre-synthesis programmable resolution of Fe(II) grid complexes, we designed and synthesized two novel intrinsically chiral ligands by appending chiral moieties to the parent ligand. The complexation of these chiral ligands with Fe(II) salt resulted in the formation of enantiomerically pure Fe(II) grid complexes, as unambiguously elucidated by CD and XRD studies. The enantiomeric complexes exhibited similar gradual and half-complete thermal and photo-induced SCO characteristics. The good agreement between the experimentally obtained and calculated CD spectra further supports the enantiomeric purity of the complexes and even the magnetic studies. The chiral resolution of Fe(II)- [2×2] grid complexes reported in this study, for the first time, might enable the fabrication of magneto-chiral molecular devices.
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http://dx.doi.org/10.1002/chem.202101432DOI Listing
June 2021

Overlapping Properties of the Short Membrane-Active Peptide BP100 With (i) Polycationic TAT and (ii) α-helical Magainin Family Peptides.

Front Cell Infect Microbiol 2021 26;11:609542. Epub 2021 Apr 26.

Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.

BP100 is a short, designer-made membrane-active peptide with multiple functionalities: antimicrobial, cell-penetrating, and fusogenic. Consisting of five lysines and 6 hydrophobic residues, BP100 was shown to bind to lipid bilayers as an amphipathic α-helix, but its mechanism of action remains unclear. With these features, BP100 embodies the characteristics of two distinctly different classes of membrane-active peptides, which have been studied in detail and where the mechanism of action is better understood. On the one hand, its amphiphilic helical structure is similar to the pore forming magainin family of antimicrobial peptides, though BP100 is much too short to span the membrane. On the other hand, its length and high charge density are reminiscent of the HIV-TAT family of cell penetrating peptides, for which inverted micelles have been postulated as translocation intermediates, amongst other mechanisms. Assays were performed to test the antimicrobial and hemolytic activity, the induced leakage and fusion of lipid vesicles, and cell uptake. From these results the functional profiles of BP100, HIV-TAT, and the magainin-like peptides magainin 2, PGLa, MSI-103, and MAP were determined and compared. It is observed that the activity of BP100 resembles most closely the much longer amphipathic α-helical magainin-like peptides, with high antimicrobial activity along with considerable fusogenic and hemolytic effects. In contrast, HIV-TAT shows almost no antimicrobial, fusogenic, or hemolytic effects. We conclude that the amphipathic helix of BP100 has a similar membrane-based activity as magainin-like peptides and may have a similar mechanism of action.
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http://dx.doi.org/10.3389/fcimb.2021.609542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107365PMC
June 2021

Structural and functional characterization of the pore-forming domain of pinholin S68.

Proc Natl Acad Sci U S A 2020 11 5;117(47):29637-29646. Epub 2020 Nov 5.

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, 76021 Karlsruhe, Germany;

Pinholin S68 triggers the lytic cycle of bacteriophage φ21 in infected Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual "pinholes." Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter ( = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (GxxxSxxxG) together with a patch of H-bonding residues (T, T, N) sideways along TMD2, available for helix-helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).
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http://dx.doi.org/10.1073/pnas.2007979117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703622PMC
November 2020

Phosphate-dependent aggregation of [KL] peptides affects their membranolytic activity.

Sci Rep 2020 07 23;10(1):12300. Epub 2020 Jul 23.

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology (KIT), POB 3640, 76021, Karlsruhe, Germany.

In this study, we investigate how the length of amphiphilic β-sheet forming peptides affects their interaction with membranes. Four polycationic model peptides with lengths from 6 to 18 amino acids were constructed from simple Lys-Leu repeats, giving [KL]. We found that (1) they exhibit a pronounced antimicrobial activity with an intriguing length dependent maximum for [KL] with 10 amino acids; (2) their hemolytic effect, on the other hand, increases steadily with peptide length. CD analysis (3) and TEM (4) show that all peptides-except for the short [KL]-aggregate into amyloid-like fibrils in the presence of phosphate ions, which in turn has a critical effect on the results in (1) and (2). In fact, (5) vesicle leakage reveals an intrinsic membrane-perturbing activity (at constant peptide mass) of [KL] > [KL] > [KL] in phosphate buffer, which changes to [KL] ≈ [KL] ≈ [KL] in PIPES. A specific interaction with phosphate ions thus explains the subtle balance between two counteracting effects: phosphate-induced unproductive pre-aggregation in solution versus monomeric membrane binding and vigorous lipid perturbation due to self-assembly of the bound peptides within the bilayer. This knowledge can now be used to control and optimize the peptides in further applications.
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http://dx.doi.org/10.1038/s41598-020-69162-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378186PMC
July 2020

Diarylethene moiety as an enthalpy-entropy switch: photoisomerizable stapled peptides for modulating p53/MDM2 interaction.

Org Biomol Chem 2020 07;18(28):5359-5369

Taras Shevchenko National University of Kyiv, Vul. Volodymyrska 60, 01601 Kyiv, Ukraine and Lumobiotics GmbH, Auer Str. 2, 76227, Karlsruhe, Germany.

Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein-protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the "open" configuration, but up to eight times larger for the corresponding "closed" isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the "closed" isomers is mostly enthalpy-driven, whereas the "open" photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.
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http://dx.doi.org/10.1039/d0ob00831aDOI Listing
July 2020

Enhancing the activity of membrane remodeling epsin-peptide by trimerization.

Bioorg Med Chem Lett 2020 06 15;30(12):127190. Epub 2020 Apr 15.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address:

Modulating the structural dynamics of biomembranes by inducing bilayer curvature and lipid packing defects has been highlighted as a practical tool to modify membrane-dependent cellular processes. Previously, we have reported on an amphipathic helical peptide derived from the N-terminal segment (residues 1-18, EpN18) of epsin-1, which can promote membrane remodeling including lipid packing defects in cell membranes. However, a high concentration is required to exhibit a pronounced effect. In this study, we demonstrate a significant increase in the membrane-remodeling effect of EpN18 by constructing a branched EpN18 homotrimer. Both monomer and trimer could enhance cell internalization of octaarginine (R8), a cell-penetrating peptide. The EpN18 trimer, however, promoted the uptake of R8 at an 80-fold lower concentration than the monomer. Analysis of the generalized polarization of a polarity-sensitive dye (di-4-ANEPPDHQ) revealed a higher efficacy of trimeric EpN18 in loosening the lipid packing in the cell membrane. Circular dichroism measurements in the presence of lipid vesicles showed that the EpN18 trimer has a higher α-helix content compared with the monomer. The stronger ability of the EpN18 trimer to impede negative bilayer curvature is also corroborated by solid-state P NMR spectroscopy. Hence, trimerizing peptides can be considered a promising approach for an exponential enhancement of their membrane-remodeling performance.
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http://dx.doi.org/10.1016/j.bmcl.2020.127190DOI Listing
June 2020

Chiral supramolecular architecture of stable transmembrane pores formed by an α-helical antibiotic peptide in the presence of lyso-lipids.

Sci Rep 2020 03 13;10(1):4710. Epub 2020 Mar 13.

Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), POB 3640, 76021, Karlsruhe, Germany.

The amphipathic α-helical antimicrobial peptide MSI-103 (aka KIA21) can form stable transmembrane pores when the bilayer takes on a positive spontaneous curvature, e.g. by the addition of lyso-lipids. Solid-state P- and N-NMR demonstrated an enrichment of lyso-lipids in these toroidal wormholes. Anionic lyso-lipids provided additional stabilization by electrostatic interactions with the cationic peptides. The remaining lipid matrix did not affect the nature of the pore, as peptides maintained the same orientation independent of lipid charge, and a change in membrane thickness did not considerably affect their tilt angle. Under optimized conditions (i.e. in the presence of lyso-lipids and appropriate bilayer thickness), stable and well-aligned pores could be obtained for solid-state H-NMR analysis. These data revealed for the first time the complete 3D alignment of this representative amphiphilic peptide in fluid membranes, which is compatible with either monomeric helices as constituents, or left-handed supercoiled dimers as building blocks from which the overall toroidal wormhole is assembled.
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http://dx.doi.org/10.1038/s41598-020-61526-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070102PMC
March 2020

Terminal charges modulate the pore forming activity of cationic amphipathic helices.

Biochim Biophys Acta Biomembr 2020 04 29;1862(4):183243. Epub 2020 Feb 29.

Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), POB 3640, 76021 Karlsruhe, Germany; KIT, Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany. Electronic address:

KIA peptides are a series of designer-made cationic amphipathic α-helical antimicrobial peptides of different lengths, based on the repetitive sequence [KIAGKIA]. They can form toroidal pores in membranes, wherein the helices are aligned in a transmembrane orientation. Solid-state N NMR is used here to differentiate between the surface-bound and transmembrane states. We find that the pore-forming activity increases when the peptides carry a positive charge (Lys residue) at the N-terminus, compared to a hydrophobic Ile-Ala N-terminal motif. In contrast, a positive charge at the C-terminus gives a lower membrane activity compared to C-terminal Ile-Ala. For peptides with otherwise identical sequence, a more than ten-fold difference in vesicle leakage can be observed, depending on which terminus carries the charge. This difference is attributed to a shift in the equilibrium between peptide helices oriented on the membrane surface and those inserted into the membrane in a pore-forming state. We show that the 3D hydrophobic moment can be used to predict which peptide sequence is more prone to form pores and will thereby show a higher membranolytic activity.
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http://dx.doi.org/10.1016/j.bbamem.2020.183243DOI Listing
April 2020

Light-controllable dithienylethene-modified cyclic peptides: photoswitching the in vivo toxicity in zebrafish embryos.

Beilstein J Org Chem 2020 7;16:39-49. Epub 2020 Jan 7.

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology (KIT), POB 3640, 76021 Karlsruhe, Germany.

This study evaluates the embryotoxicity of dithienylethene-modified peptides upon photoswitching, using 19 analogues based on the β-hairpin scaffold of the natural membranolytic peptide gramicidin S. We established an in vivo assay in two variations (with ex vivo and in situ photoisomerization), using larvae of the model organism and determined the toxicities of the peptides in terms of 50% lethal doses (LD). This study allowed us to: (i) demonstrate the feasibility of evaluating peptide toxicity with larvae at 3-4 days post fertilization, (ii) determine the phototherapeutic safety windows for all peptides, (iii) demonstrate photoswitching of the whole-body toxicity for the dithienylethene-modified peptides in vivo, (iv) re-analyze previous structure-toxicity relationship data, and (v) select promising candidates for potential clinical development.
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http://dx.doi.org/10.3762/bjoc.16.6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964649PMC
January 2020

Monofluoroalkene-Isostere as a F NMR Label for the Peptide Backbone: Synthesis and Evaluation in Membrane-Bound PGLa and (KIGAKI).

Chemistry 2020 Feb 23;26(7):1511-1517. Epub 2020 Jan 23.

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, P. O. Box 3640, 76021, Karlsruhe, Germany.

Solid-state F NMR is a powerful method to study the interactions of biologically active peptides with membranes. So far, in labelled peptides, the F-reporter group has always been installed on the side chain of an amino acid. Given the fact that monofluoroalkenes are non-hydrolyzable peptide bond mimics, we have synthesized a monofluoroalkene-based dipeptide isostere, Val-Ψ[(Z)-CF=CH]-Gly, and inserted it in the sequence of two well-studied antimicrobial peptides: PGLa and (KIGAKI) are representatives of an α-helix and a β-sheet. The conformations and biological activities of these labeled peptides were studied to assess the suitability of monofluoroalkenes for F NMR structure analysis.
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http://dx.doi.org/10.1002/chem.201905054DOI Listing
February 2020

Shape-Memory Effect by Sequential Coupling of Functions over Different Length Scales in an Architectured Hydrogel.

Biomacromolecules 2020 02 14;21(2):680-687. Epub 2020 Jan 14.

Institute of Biomaterial Science , Helmholtz-Zentrum Geesthacht , Kantstraße. 55 , 14513 Teltow , Germany.

The integration of functions in materials in order to gain macroscopic effects in response to environmental changes is an ongoing challenge in material science. Here, functions on different hierarchical levels are sequentially linked to translate a pH-triggered conformational transition from the molecular to the macroscopic level to induce directed movements in hydrogels. When the pH is increased, lysine-rich peptide molecules change their conformation into a β-hairpin structure because of the reduced electrostatic repulsion among the deprotonated amino groups. Coupled to this conformation change is the capability of the β-hairpin motifs to subsequently assemble into aggregates acting as reversible cross-links, which are used as controlling units to fix a temporary macroscopic shape. A structural function implemented into the hydrogel by a microporous architecture-enabled nondisruptive deformation upon compression by buckling of pore walls and their elastic recovery. Coupled to this structural function is the capability of the porous material to enhance the diffusion of ions into the hydrogel and to keep the dimension of the macroscopic systems almost constant when the additional cross-links are formed or cleaved as it limits the dimensional change of the pore walls. Covalent cross-linking of the hydrogel into a polymer network acted as gear shift to ensure translation of the function on the molecular level to the macroscopic dimension. In this way, the information of a directed shape-shift can be programmed into the material by mechanical deformation and pH-dependent formation of temporary net points. The information could be read out by lowering the pH. The peptides reverted back into their original random coil conformation and the porous polymer network could recover from the previously applied elastic deformation. The level of multifunctionality of the hydrogels can be increased by implementation of additional orthogonal functions such as antimicrobicity by proper selection of multifunctional peptides, which could enable sophisticated biomedical devices.
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http://dx.doi.org/10.1021/acs.biomac.9b01390DOI Listing
February 2020

Supreme activity of gramicidin S against resistant, persistent and biofilm cells of staphylococci and enterococci.

Sci Rep 2019 11 29;9(1):17938. Epub 2019 Nov 29.

Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry (IOC), Karlsruhe, 76131, Germany.

Three promising antibacterial peptides were studied with regard to their ability to inhibit the growth and kill the cells of clinical strains of Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. The multifunctional gramicidin S (GS) was the most potent, compared to the membranotropic temporin L (TL), being more effective than the innate-defence regulator IDR-1018 (IDR). These activities, compared across 16 strains as minimal bactericidal and minimal inhibitory concentrations (MIC), are independent of bacterial resistance pattern, phenotype variations and/or biofilm-forming potency. For S. aureus strains, complete killing is accomplished by all peptides at 5 × MIC. For E. faecalis strains, only GS exhibits a rapid bactericidal effect at 5 × MIC, while TL and IDR require higher concentrations. The biofilm-preventing activities of all peptides against the six strains with the largest biofilm biomass were compared. GS demonstrates the lowest minimal biofilm inhibiting concentrations, whereas TL and IDR are consistently less effective. In mature biofilms, only GS completely kills the cells of all studied strains. We compare the physicochemical properties, membranolytic activities, model pharmacokinetics and eukaryotic toxicities of the peptides and explain the bactericidal, antipersister and antibiofilm activities of GS by its elevated stability, pronounced cell-penetration ability and effective utilization of multiple modes of antibacterial action.
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http://dx.doi.org/10.1038/s41598-019-54212-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884456PMC
November 2019

Bilayer thickness determines the alignment of model polyproline helices in lipid membranes.

Phys Chem Chem Phys 2019 Oct;21(40):22396-22408

Institute of Chemistry, Technical University of Berlin, Müller-Breslau-Str. 10, Berlin 10623, Germany and Department of Chemistry, University of Manitoba, Dysart Rd. 144, Winnipeg MB R3T 2N2, Canada.

Our understanding of protein folds relies fundamentally on the set of secondary structures found in the proteomes. Yet, there also exist intriguing structures and motifs that are underrepresented in natural biopolymeric systems. One example is the polyproline II helix, which is usually considered to have a polar character and therefore does not form membrane spanning sections of membrane proteins. In our work, we have introduced specially designed polyproline II helices into the hydrophobic membrane milieu and used 19F NMR to monitor the helix alignment in oriented lipid bilayers. Our results show that these artificial hydrophobic peptides can adopt several different alignment states. If the helix is shorter than the thickness of the hydrophobic core of the membrane, it is submerged into the bilayer with its long axis parallel to the membrane plane. The polyproline helix adopts a transmembrane alignment when its length exceeds the bilayer thickness. If the peptide length roughly matches the lipid thickness, a coexistence of both states is observed. We thus show that the lipid thickness plays a determining role in the occurrence of a transmembrane polyproline II helix. We also found that the adaptation of polyproline II helices to hydrophobic mismatch is in some notable aspects different from α-helices. Finally, our results prove that the polyproline II helix is a competent structure for the construction of transmembrane peptide segments, despite the fact that no such motif has ever been reported in natural systems.
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http://dx.doi.org/10.1039/c9cp02996fDOI Listing
October 2019

4-Aminophthalimide Amino Acids as Small and Environment-Sensitive Fluorescent Probes for Transmembrane Peptides.

Chembiochem 2020 03 12;21(5):618-622. Epub 2019 Nov 12.

Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131, Karlsruhe, Germany.

Fluorescence probing of transmembrane (TM) peptides is needed to complement state-of-the art methods-mainly oriented circular dichroism and solid-state NMR spectroscopy-and to allow imaging in living cells. Three new amino acids incorporating the solvatofluorescent 4-aminophthalimide in their side chains were synthesized in order to examine the local polarity in the α-helical TM fragment of the human epidermal growth factor receptor. It was possible to distinguish their locations, either in the hydrophobic core of the lipid bilayer or at the membrane surface, by fluorescence readout, including blue shift and increased quantum yield. An important feature is the small size of the 4-aminophthalimide chromophore. It makes one of the new amino acids approximately isosteric to tryptophan, typically used as a very small fluorescent amino acid in peptides and proteins. In contrast to the only weakly fluorescent indole system in tryptophan, the 4-aminophthalimide moiety produces a significantly more informative fluorescence readout and is selectively excited outside the biopolymer absorption range.
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http://dx.doi.org/10.1002/cbic.201900520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079057PMC
March 2020

Inhibition of Pseudomonas aeruginosa biofilm formation and expression of virulence genes by selective epimerization in the peptide Esculentin-1a(1-21)NH.

FEBS J 2019 10 13;286(19):3874-3891. Epub 2019 Jun 13.

Laboratory affiliated to Pasteur Italia-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Italy.

Pseudomonas aeruginosa is a pathogenic bacterium known to cause serious human infections, especially in immune-compromised patients. This is due to its unique ability to transform from a drug-tolerant planktonic to a more dangerous and treatment-resistant sessile life form, called biofilm. Recently, two derivatives of the frog skin antimicrobial peptide esculentin-1a, i.e. Esc(1-21) and its D-amino acids containing diastereomer Esc(1-21)-1c, were characterized for their powerful anti-Pseudomonal activity against both forms. Prevention of biofilm formation already in its early stages could be even more advantageous for counteracting infections induced by this bacterium. In this work, we studied how the diastereomer Esc(1-21)-1c can inhibit Pseudomonas biofilm formation in comparison to the parent peptide and two clinically-used conventional antibiotics, i.e. colistin and aztreonam, when applied at dosages below the minimal growth inhibitory concentration. Biofilm prevention was correlated to the peptides' ability to inhibit Pseudomonas motility and to reduce the production of virulent metabolites, for example, pyoverdine and rhamnolipids. Furthermore, the molecular mechanism underlying these activities was evaluated by studying the peptides' effect on the expression of key genes involved in the virulence and motility of bacteria, as well as by monitoring the peptides' binding to the bacterial signaling nucleotide ppGpp. Our results demonstrate that the presence of only two D-amino acids in Esc(1-21)-1c is sufficient to downregulate ppGpp-mediated expression of biofilm-associated genes, presumably as a result of higher peptide stability and therefore prolonged interaction with the nucleotide. Overall, these studies should assist efficient design and optimization of new anti-infective agents with multiple pharmacologically beneficial properties.
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http://dx.doi.org/10.1111/febs.14940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779485PMC
October 2019

Biocompatibility of Amine-Functionalized Silica Nanoparticles: The Role of Surface Coverage.

Small 2019 03 5;15(10):e1805400. Epub 2019 Feb 5.

Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Campus North, Hermann-von-Helmholtz-Platz 1, D-76344, Eggenstein-Leopoldshafen, Germany.

Here, amorphous silica nanoparticles (NPs), one of the most abundant nanomaterials, are used as an example to illustrate the utmost importance of surface coverage by functional groups which critically determines biocompatibility. Silica NPs are functionalized with increasing amounts of amino groups, and the number of surface exposed groups is quantified and characterized by detailed NMR and fluorescamine binding studies. Subsequent biocompatibility studies in the absence of serum demonstrate that, irrespective of surface modification, both plain and amine-modified silica NPs trigger cell death in RAW 264.7 macrophages. The in vitro results can be confirmed in vivo and are predictive for the inflammatory potential in murine lungs. In the presence of serum proteins, on the other hand, a replacement of only 10% of surface-active silanol groups by amines is sufficient to suppress cytotoxicity, emphasizing the relevance of exposure conditions. Mechanistic investigations identify a key role of lysosomal injury for cytotoxicity only in the presence, but not in the absence, of serum proteins. In conclusion, this work shows the critical need to rigorously characterize the surface coverage of NPs by their constituent functional groups, as well as the impact of serum, to reliably establish quantitative nanostructure activity relationships and develop safe nanomaterials.
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http://dx.doi.org/10.1002/smll.201805400DOI Listing
March 2019

Tetrameric Charge-Zipper Assembly of the TisB Peptide in Membranes-Computer Simulation and Experiment.

J Phys Chem B 2019 02 14;123(8):1770-1779. Epub 2019 Feb 14.

Institute of Biological Interfaces (IBG-2) , Karlsruhe Institute of Technology , P. O. Box 3640 , 76021 Karlsruhe , Germany.

TisB is a short amphiphilic α-helical peptide from Escherichia coli that induces a breakdown of the pH gradient across the inner membrane when the bacteria are under stress and require to form persister cells to turn into a biofilm. A computational-experimental approach combining all-atom and coarse-grained molecular dynamics simulation with circular dichroism spectroscopy and gel electrophoresis was used to reveal its structure and oligomeric assembly in a phospholipid bilayer. TisB is found to be inserted upright in the membrane as a tetrameric bundle with a left-handed sense of supercoiling, best described as an antiparallel dimer-of-dimers. The tetramer is stabilized by means of a regular but dynamically interchanging pattern of salt bridges and hydrogen bonds, in accordance with the recently proposed "charge-zipper" motif.
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http://dx.doi.org/10.1021/acs.jpcb.8b12087DOI Listing
February 2019

Molecular structure and function of myelin protein P0 in membrane stacking.

Sci Rep 2019 01 24;9(1):642. Epub 2019 Jan 24.

Department of Biomedicine, University of Bergen, Bergen, Norway.

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in changes of the bilayer properties, but also potentially involves the arrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.
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http://dx.doi.org/10.1038/s41598-018-37009-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345808PMC
January 2019

Structure-Activity Relationships of Photoswitchable Diarylethene-Based β-Hairpin Peptides as Membranolytic Antimicrobial and Anticancer Agents.

J Med Chem 2018 12 4;61(23):10793-10813. Epub 2018 Dec 4.

Institute of Biological Interfaces (IBG-2) , Karlsruhe Institute of Technology (KIT) , POB 3640, 76021 Karlsruhe , Germany.

Five series (28 structures) of photoswitchable β-hairpin peptides were synthesized based on the cyclic scaffold of the natural antibiotic gramicidin S. Cell-type selectivity was compared for all activated (diarylethene "ring-open") and deactivated ("ring-closed") forms in terms of antibacterial activity (MIC against Escherichia coli and Bacillus subtilis), anticancer activity (IC against HeLa cell line), and hemolytic cytotoxicity (HC against human erythrocytes). Correlations between the conformational plasticity of the peptides, their hydrophobicity, and their bioactivity were also analyzed. Considerable improvements in selectivity were achieved compared to the reference compound. We found a dissociation of the anticancer activity from hemolysis. Phototherapeutic indices (PTI), HC(closed)/MIC(open) and HC(closed)/IC(open), were introduced for the peptides as safety criteria. The highest PTI for HeLa-selective toxicity were observed among analogues containing hydroxyleucine on the hydrophobic face. For one compound, high PTIs were demonstrated across a range of different cancer cell lines, including a doxorubicin-resistant one.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01428DOI Listing
December 2018

Highly reactive bis-cyclooctyne-modified diarylethene for SPAAC-mediated cross-linking.

Org Biomol Chem 2018 11;16(44):8559-8564

University Chemical Laboratory, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, UK.

Photoisomerizable diarylethenes equipped with triple bonds are promising building blocks for constructing bistable photocontrollable systems. Here we report on the design, synthesis and application of a cross-linking reagent which is based on a diarylethene core and features two strained cyclooctynes. High reactivity of the cyclooctyne rings in catalyst-free 1,3-dipolar cycloaddition reactions was suggested to stem from the additional strain imposed by the fused thiophene rings. This hypothesis was confirmed by quantum chemical calculations.
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http://dx.doi.org/10.1039/c8ob02428fDOI Listing
November 2018

Best of Two Worlds? How MD Simulations of Amphiphilic Helical Peptides in Membranes Can Complement Data from Oriented Solid-State NMR.

J Chem Theory Comput 2018 Nov 5;14(11):6002-6014. Epub 2018 Oct 5.

Institute of Organic Chemistry , Karlsruhe Institute of Technology (KIT) , Fritz-Haber-Weg 6 , 76131 Karlsruhe , Germany.

The membrane alignment of helical amphiphilic peptides in oriented phospholipid bilayers can be obtained as ensemble and time averages from solid state H NMR by fitting the quadrupolar splittings to ideal α-helices. At the same time, molecular dynamics (MD) simulations can provide atomistic insight into peptide-membrane systems. Here, we evaluate the potential of MD simulations to complement the experimental NMR data that is available on three exemplary systems: the natural antimicrobial peptide PGLa and the two designer-made peptides MSI-103 and KIA14, whose sequences were derived from PGLa. Each peptide was simulated for 1 μs in a DMPC lipid bilayer. We calculated from the MD simulations the local angles which define the side chain geometry with respect to the peptide helix. The peptide orientation was then calculated (i) directly from the simulation, (ii) from back-calculated MD-derived NMR splittings, and (iii) from experimental H NMR splittings. Our findings are that (1) the membrane orientation and secondary structure of the peptides found in the NMR analysis are generally well reproduced by the simulations; (2) the geometry of the side chains with respect to the helix backbone can deviate significantly from the ideal structure depending on the specific residue, but on average all side chains have the same orientation; and (3) for all of our peptides, the azimuthal rotation angle found from the MD-derived splittings is about 15° smaller than the experimental value.
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http://dx.doi.org/10.1021/acs.jctc.8b00283DOI Listing
November 2018

Protein ORIGAMI: A program for the creation of 3D paper models of folded peptides.

Biochem Mol Biol Educ 2018 07 8;46(4):403-409. Epub 2018 Jul 8.

Institute of Organic Chemistry (IOC), Karlsruhe Institute of Technology (KIT), Karlsruhe, 76133, Germany.

Protein ORIGAMI (http://ibg.kit.edu/protein_origami) is a browser-based web application that allows the user to create straightforward 3D paper models of folded peptides for research, teaching and presentations. An amino acid sequence can be turned into α-helices, β-strands and random coils that can be printed out and folded into properly scaled models, with a color code denoting the biophysical characteristics of each amino acid residue (hydrophobicity, charge, etc.). These models provide an intuitive visual and tactile understanding of peptide interactions with other partners, such as helix-helix assembly, oligomerization, membrane binding, or pore formation. Helices can also be displayed as a helical wheel or helical mesh in 2D graphics, to be used in publications or presentations. The highly versatile programme Protein ORIGAMI is also suited to create less conventional helices with arbitrary pitch (e.g., 3 -helix, π-helix, or left-handed helices). Noncanonical amino acids, labels and different terminal modifications can be defined and displayed at will, and different protonation states can be shown. In addition to the web application, the program source code can be downloaded and installed locally on a PC. The printed paper models can be readily used for daily research and discussions, just as for educational purposes and teaching. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:403-409, 2018.
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http://dx.doi.org/10.1002/bmb.21132DOI Listing
July 2018

Helix Fraying and Lipid-Dependent Structure of a Short Amphipathic Membrane-Bound Peptide Revealed by Solid-State NMR.

J Phys Chem B 2018 06 1;122(23):6236-6250. Epub 2018 Jun 1.

Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2) , P.O. Box 3640, 76021 Karlsruhe , Germany.

The amphipathic α-helical peptide KIA14 [(KIAGKIA)-NH] was studied in membranes using circular dichroism and solid-state NMR spectroscopy to obtain global as well as local structural information. By analyzing H NMR data from 10 analogues of KIA14 that were selectively labeled with Ala- d, those positions that are properly folded into a helix could be determined within the membrane-bound peptide. The N-terminus was found to be unraveled, whereas positions 4-14 formed an ideal helix all the way to the C-terminus. The helicity did not change when Gly residues were replaced by Ala- d but was reduced when Ile was replaced, indicating that large hydrophobic residues are required for membrane binding and helix formation. The reduced helicity was strongly correlated with a decrease in peptide-induced leakage from lipid vesicles. The orientation of the short KIA14 peptide was assessed in several lipid systems and compared with that of the longer KIA21 sequence [(KIAGKIA)-NH]. In 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine, both peptides are aligned flat on the membrane surface, whereas in 1,2-dimyristoyl- sn-glycero-3-phosphatidylcholine (DMPC)/1-myristoyl-2-hydroxy- sn-glycero-3-phosphatidylcholine (lyso-MPC) both are inserted into the membrane in an upright orientation. These two types of lipid systems had been selected for their strongly negative and positive spontaneous curvature, respectively. We propose that in these cases, the peptide orientation is largely determined by the lipid properties. On the other hand, in plain DMPC and 1,2-dilauroyl- sn-glycero-3-phosphatidylcholine, which have only a slight positive curvature, a marked difference in orientation is evident: the short KIA14 lies almost flat on the membrane surface, whereas the longer KIA21 is more tilted. We thus propose that out of the lipid systems tested here, DMPC (with hardly any curvature) is the least biased lipid system in which peptide orientation and realignment can be studied, allowing to compare and discriminate the intrinsic effects of the properties of the peptides as such.
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http://dx.doi.org/10.1021/acs.jpcb.8b02661DOI Listing
June 2018

Transmembrane Polyproline Helix.

J Phys Chem Lett 2018 May 13;9(9):2170-2174. Epub 2018 Apr 13.

Institute of Chemistry , Technical University of Berlin , Müller-Breslau-Strasse 10 , Berlin 10623 , Germany.

The third most abundant polypeptide conformation in nature, the polyproline-II helix, is a polar, extended secondary structure with a local organization stabilized by intercarbonyl interactions within the peptide chain. Here we design a hydrophobic polyproline-II helical peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold and demonstrate its transmembrane alignment in model lipid bilayers by means of solid-state F NMR. As result, we provide a first example of a purely artificial transmembrane peptide with a structural organization that is not based on hydrogen-bonding.
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http://dx.doi.org/10.1021/acs.jpclett.8b00829DOI Listing
May 2018

Efficiently Photocontrollable or Not? Biological Activity of Photoisomerizable Diarylethenes.

Chemistry 2018 Aug 3;24(44):11245-11254. Epub 2018 Jul 3.

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, POB 3640, 76021, Karlsruhe, Germany.

Diarylethene derivatives, the biological activity of which can be reversibly changed by irradiation with light of different wavelengths, have shown promise as scientific tools and as candidates for photocontrollable drugs. However, examples demonstrating efficient photocontrol of their biological activity are still relatively rare. This concept article discusses the possible reasons for this situation and presents a critical analysis of existing data and hypotheses in this field, in order to extract the design principles enabling the construction of efficient photocontrollable diarylethene-based molecules. Papers addressing biologically relevant interactions between diarylethenes and biomolecules are analyzed; however, in most published cases, the efficiency of photocontrol in living systems remains to be demonstrated. We hope that this article will encourage further discussion of design principles, primarily among pharmacologists, synthetic and medicinal chemists.
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http://dx.doi.org/10.1002/chem.201801205DOI Listing
August 2018

Roles of Amphipathicity and Hydrophobicity in the Micelle-Driven Structural Switch of a 14-mer Peptide Core from a Choline-Binding Repeat.

Chemistry 2018 Apr 15;24(22):5825-5839. Epub 2018 Mar 15.

Instituto de Química Física Rocasolano (IQFR), Consejo Superior de Investigaciones Científicas (CSIC), Serrano 119, 28006, Madrid, Spain.

Choline-binding repeats (CBRs) are ubiquitous sequences with a β-hairpin core that are found in the surface proteins of several microorganisms such as S. pneumoniae (pneumococcus). Previous studies on a 14-mer CBR sequence derived from the pneumoccal LytA autolysin (LytA peptide) have demonstrated a switch behaviour for this peptide, so that it acquires a stable, native-like β-hairpin conformation in aqueous solution but is reversibly transformed into an amphipathic α-helix in the presence of detergent micelles. With the aim of understanding the factors responsible for this unusual β-hairpin to α-helix transition, and to specifically assess the role of peptide hydrophobicity and helical amphipathicity in the process, we designed a series of LytA variants affecting these two parameters and studied their interaction with dodecylphosphocholine (DPC) micelles by solution NMR, circular dichroism and fluorescence spectroscopies. Our results indicate that stabilising cross-strand interactions become essential for β-hairpin stability in the absence of optimal turn sequences. Moreover, both amphipathicity and hydrophobicity display comparable importance for helix stabilisation of CBR-derived peptides in micelles, indicating that these sequences represent a novel class of micelle/membrane-interacting peptides.
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http://dx.doi.org/10.1002/chem.201704802DOI Listing
April 2018

Orthogonal F-Labeling for Solid-State NMR Spectroscopy Reveals the Conformation and Orientation of Short Peptaibols in Membranes.

Chemistry 2018 Mar 21;24(17):4328-4335. Epub 2018 Feb 21.

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology (KIT), POB 3640, 76021, Karlsruhe, Germany.

Peptaibols are promising drug candidates in view of their interference with cellular membranes. Knowledge of their lipid interactions and membrane-bound structure is needed to understand their activity and should be, in principle, accessible by solid-state NMR spectroscopy. However, their unusual amino acid composition and noncanonical conformations make it very challenging to find suitable labels for NMR spectroscopy. Particularly in the case of short sequences, new strategies are required to maximize the structural information that can be obtained from each label. Herein, l-3-(trifluoromethyl)bicyclopent[1.1.1]-1-ylglycine, (R)- and (S)-trifluoromethylalanine, and N-backbone labels, each probing a different direction in the molecule, have been combined to elucidate the conformation and membrane alignment of harzianin HK-VI. For the short sequence of 11 amino acids, 12 orientational constraints have been obtained by using F and N NMR spectroscopy. This strategy revealed a β-bend ribbon structure, which becomes realigned in the membrane from a surface-parallel state towards a membrane-spanning state, with increasing positive spontaneous curvature of the lipids.
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http://dx.doi.org/10.1002/chem.201704307DOI Listing
March 2018
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