Publications by authors named "Jochen Bürck"

62 Publications

Chirality Remote Control in Nanoporous Materials by Circularly Polarized Light.

J Am Chem Soc 2021 May 29;143(18):7059-7068. Epub 2021 Apr 29.

Institute of Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.

The ability to dynamically control chirality remains a grand challenge in chemistry. Although many molecules possess chiral isomers, lacking their isolation, for instance during photoisomerization, results in racemic mixtures with suppressed enantiospecific chiral properties. Here, we present a nanoporous solid in which chirality and enantioselective enrichment is induced by circularly polarized light (CPL). The material is based on photoswitchable fluorinated azobenzenes attached to the scaffold of a crystalline metal-organic framework (MOF). The azobenzene undergoes -to--photoisomerization upon irradiation with green light and reverts back to upon violet light. While each moiety in conformation is chiral, we show the isomer also possesses a nonplanar, chiral conformation. During photoisomerization with unpolarized light, no enantiomeric enrichment is observed and both isomers, - and - as well as and -, respectively, are formed in identical quantities. In contrast, CPL causes chiral photoresolution, resulting in an optically active material. Right-CPL selectively excites - and - enantiomers, producing a MOF with enriched -enantiomers, and . The induction of optical activity is reversible and only depends on the light-handedness. As shown by first-principle DFT calculations, while both, and , are stabilized in nonplanar, chiral conformations in the MOF, the isomer adopts a planar, achiral form in solution, as verified experimentally. This shows that the chiral photoresolution is enabled by the linker reticulation in the MOF. Our study demonstrates the induction of chirality and optical activity in solid materials by CPL and opens new opportunities for chiral resolution and information storage with CPL.
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http://dx.doi.org/10.1021/jacs.1c01693DOI Listing
May 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

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

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

Switching the enantioselectivity of nanoporous host materials by light.

Chem Commun (Camb) 2019 Jul;55(60):8776-8779

Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces (IFG), 76344 Eggenstein-Leopoldshafen, Germany.

A chiral photoswitchable nanoporous material with remote-controllable enantioselective adsorption capacity is presented. This metal-organic framework possesses both homochiral d-camphoric acid and light-responsive azobenzene moieties. Although the structure at the chiral moieties is unaffected, the trans-cis-azobenzene-photoisomerization changes the pore environment and, thus, switches the enantioselective adsorption behavior of the homochiral MOF.
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http://dx.doi.org/10.1039/c9cc02849hDOI Listing
July 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

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

Molecular mechanism of synergy between the antimicrobial peptides PGLa and magainin 2.

Sci Rep 2017 10 13;7(1):13153. Epub 2017 Oct 13.

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

PGLa and magainin 2 (MAG2) are amphiphilic α-helical membranolytic peptides from frog skin with known synergistic antimicrobial activity. By systematically mutating residues in the two peptides it was possible to identify the ones crucial for the synergy, as monitored by biological assays, fluorescence vesicle leakage, and solid-state N-NMR. Electrostatic interactions between anionic groups in MAG2 and cationic residues in PGLa enhance synergy but are not necessary for the synergistic effect. Instead, two Gly residues (7 and 11) in a so-called GxxxG motif in PGLa are necessary for synergy. Replacing either of them with Ala or another hydrophobic residue completely abolishes synergy according to all three methods used. The designer-made peptide MSI-103, which has a similar sequence as PGLa, shows no synergy with MAG2, but by introducing two Gly mutations it was possible to make it synergistic. A molecular model is proposed for the functionally active PGLa-MAG2 complex, consisting of a membrane-spanning antiparallel PGLa dimer that is stabilized by intimate Gly-Gly contacts, and where each PGLa monomer is in contact with one MAG2 molecule at its C-terminus.
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http://dx.doi.org/10.1038/s41598-017-12599-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640672PMC
October 2017

Lactam-Stapled Cell-Penetrating Peptides: Cell Uptake and Membrane Binding Properties.

J Med Chem 2017 10 26;60(19):8071-8082. Epub 2017 Sep 26.

Institute of Biological Interfaces, Karlsruhe Institute of Technology , POB 3640, 76021 Karlsruhe, Germany.

Stapling of side chains to stabilize an α-helical structure has been generally associated with an increased uptake of CPPs. Here, we compare four amphiphilic stapled peptides with their linear counterparts in terms of their membrane binding and conformational features in order to correlate these with uptake efficiency and toxicological effects. The impact of lactam stapling was found to vary strongly with regard to the different aspects of peptide-membrane interactions. Nearly all stapled peptides caused less membrane perturbation (vesicle leakage, hemolysis, bacterial lysis) than their linear counterparts. In one case (MAP-1) where stapling enhanced α-helicity in aqueous and lipid environments, leakage was eliminated while cell uptake in HEK293 and HeLa cells remained high, which improved the overall characteristics. The other systems (DRIM, WWSP, KFGF) did not improve, however. The data suggest that cell uptake of amphipathic CPPs correlates with their adopted α-helix content in membranes rather than their helicity in solution.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00813DOI Listing
October 2017

Structure analysis of the membrane-bound dermcidin-derived peptide SSL-25 from human sweat.

Biochim Biophys Acta Biomembr 2017 Dec 6;1859(12):2308-2318. Epub 2017 Sep 6.

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

SSL-25 (SSLLEKGLDGAKKAVGGLGKLGKDA) is one of the shortest peptides present in human sweat and is produced after the proteolytic processing of the parent peptide dermcidin. Both peptides are reported to have antimicrobial function. To determine the structure of SSL-25 in lipid bilayers, a series of F-labeled SSL-25 analogs were synthesized. Circular dichroism (CD) analysis showed that SSL-25 and all of its analogs formed α-helices in the presence of lipid vesicles, thus allowing a detailed analysis via oriented CD and solid-state NMR. The results suggest that SSL-25 resides on the membrane surface with a slight helix tilt angle. A detailed F NMR analysis revealed that SSL-25 does not form a continuous helix. The α-helical structure of the N-terminal part of the peptide was preserved in membranes of different lipid compositions and at various peptide-to-lipid molar ratios, but the C-terminus was disordered and did not fold into a well-defined α-helical conformation. Furthermore, the NMR results showed that SSL-25 resides on the membrane surface and does not re-orient into the membrane in response to changes in either peptide concentration or membrane composition. SSL-25 does not aggregate and remains fully mobile within the membrane bilayer, as shown by F NMR. SSL-25 has a high binding affinity toward bilayers mimicking bacterial lipid compositions, but does not bind to mammalian model membranes containing cholesterol. These observations may explain the selectivity of this peptide for bacterial membranes, and they are also in line with basic biophysical considerations on spontaneous lipid curvature and the general effect of cholesterol on peptide/lipid interactions.
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http://dx.doi.org/10.1016/j.bbamem.2017.09.004DOI Listing
December 2017

Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line.

Sci Rep 2017 07 10;7(1):4974. Epub 2017 Jul 10.

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

Compact myelin comprises most of the dry weight of myelin, and its insulative nature is the basis for saltatory conduction of nerve impulses. The major dense line (MDL) is a 3-nm compartment between two cytoplasmic leaflets of stacked myelin membranes, mostly occupied by a myelin basic protein (MBP) phase. MBP is an abundant myelin protein involved in demyelinating diseases, such as multiple sclerosis. The association of MBP with lipid membranes has been studied for decades, but the MBP-driven formation of the MDL remains elusive at the biomolecular level. We employed complementary biophysical methods, including atomic force microscopy, cryo-electron microscopy, and neutron scattering, to investigate the formation of membrane stacks all the way from MBP binding onto a single membrane leaflet to the organisation of a stable MDL. Our results support the formation of an amorphous protein phase of MBP between two membrane bilayers and provide a molecular model for MDL formation during myelination, which is of importance when understanding myelin assembly and demyelinating conditions.
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http://dx.doi.org/10.1038/s41598-017-05364-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504075PMC
July 2017

Influence of the Length and Charge on the Activity of α-Helical Amphipathic Antimicrobial Peptides.

Biochemistry 2017 03 10;56(11):1680-1695. Epub 2017 Mar 10.

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

Hydrophobic mismatch is important for pore-forming amphipathic antimicrobial peptides, as demonstrated recently [Grau-Campistany, A., et al. (2015) Sci. Rep. 5, 9388]. A series of different length peptides have been generated with the heptameric repeat sequence KIAGKIA, called KIA peptides, and it was found that only those helices sufficiently long to span the hydrophobic thickness of the membrane could induce leakage in lipid vesicles; there was also a clear length dependence of the antimicrobial and hemolytic activities. For the original KIA sequences, the cationic charge increased with peptide length. The goal of this work is to examine whether the charge also has an effect on activity; hence, we constructed two further series of peptides with a sequence similar to those of the KIA peptides, but with a constant charge of +7 for all lengths from 14 to 28 amino acids. For both of these new series, a clear length dependence similar to that of KIA peptides was observed, indicating that charge has only a minor influence. Both series also showed a distinct threshold length for peptides to be active, which correlates directly with the thickness of the membrane. Among the longer peptides, the new series showed activities only slightly lower than those of the original KIA peptides of the same length that had a higher charge. Shorter peptides, in which Gly was replaced with Lys, showed activities similar to those of KIA peptides of the same length, but peptides in which Ile was replaced with Lys lost their helicity and were less active.
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http://dx.doi.org/10.1021/acs.biochem.6b01071DOI Listing
March 2017

Antibiotic gold: tethering of antimicrobial peptides to gold nanoparticles maintains conformational flexibility of peptides and improves trypsin susceptibility.

Biomater Sci 2017 Mar;5(4):817-827

Karlsruhe Institute of Technology (KIT), 1Institute of Biological Interfaces (IBG-2) P.O.B. 3640, D 76021 Karlsruhe, Germany. and KIT, 2Institute of Organic Chemistry & CFN, Fritz-Haber-Weg 6, D-76131 Karlsruhe, Germany.

Peptide-coated nanoparticles are valuable tools for diverse biological applications, such as drug delivery, molecular recognition, and antimicrobial action. The functionalization of pre-fabricated nanoparticles with free peptides in solution is inefficient either due to aggregation of the particles or due to the poor ligand exchange reaction. Here, we present a one-pot synthesis for preparing gold nanoparticles with a homogeneous distribution that are covered in situ with cationic peptides in a site-selective manner via Cys-residue at the N-terminus. Five representative peptides were selected, which are known to perturb cellular membranes and exert their antimicrobial and/or cell penetrating activity by folding into amphiphilic α-helical structures. When tethered to the nanoparticles at a single site, all peptides were found to switch their conformation from unordered state (in aqueous buffers) to their functionally relevant α-helical conformation in the presence of model membranes, as shown by circular dichroism spectroscopy. The conjugated peptides also maintained the same antibacterial activity as in the free form. Most importantly, when tethered to the gold nanoparticles the peptides showed an enormous increase in stability against trypsin digestion compared to the free forms, leading to a dramatic improvement of their lifetimes and activities. These findings suggest that site-selective surface tethering of peptides to gold nanoparticles has several advantages: (i) it does not prevent the peptides from folding into their biologically active conformation, (ii) such conjugation protects the peptides against protease digestion, and (iii) this way it is possible to prepare stable, water soluble antimicrobial nanoparticles as promising antibacterial agents.
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http://dx.doi.org/10.1039/c7bm00069cDOI Listing
March 2017

Homo- and heteromeric interaction strengths of the synergistic antimicrobial peptides PGLa and magainin 2 in membranes.

Eur Biophys J 2016 Sep 6;45(6):535-47. Epub 2016 Apr 6.

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

PGLa and magainin 2 (MAG2) are amphiphilic α-helical frog peptides with synergistic antimicrobial activity. In vesicle leakage assays we observed the strongest synergy for equimolar mixtures of PGLa and MAG2. This result was consistent with solid-state (15)N-NMR data on the helix alignment in model membranes. The Hill coefficients determined from the vesicle leakage data showed that the heterodimeric (PGLa-MAG2) interactions were stronger than the homodimeric (PGLa-PGLa and MAG2-MAG2) interactions. This result was also reflected in the free energy of dimerization determined from oriented circular dichroism and quantitative solid-state (19)F-NMR analysis.
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http://dx.doi.org/10.1007/s00249-016-1120-7DOI Listing
September 2016

Alanine scan and (2)H NMR analysis of the membrane-active peptide BP100 point to a distinct carpet mechanism of action.

Biochim Biophys Acta 2016 Jun 11;1858(6):1328-38. Epub 2016 Mar 11.

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

The short membrane-active peptide BP100 [KKLFKKILKYL-NH2] is known as an effective antimicrobial and cell penetrating agent. For a functional alanine scan each of the 11 amino acids was replaced with deuterated Ala-d3, one at a time. MIC assays showed that a substitution of Lys did not affect the antimicrobial activity, but it decreased when a hydrophobic residue was replaced. In most cases, a reduction in hydrophobicity led to a decrease in hemolysis, and some peptide analogues had an improved therapeutic index. Circular dichroism showed that BP100 folds as an amphiphilic α-helix in a bilayer. Its alignment was determined from (2)H NMR in oriented membranes of different composition. The azimuthal rotation angle was the same under all conditions, but the average helix tilt angle and the dynamical behavior of the peptide varied in a systematic manner. In POPC/POPG bilayers, with a negative spontaneous curvature, the peptide was found to lie flat on the bilayer surface, and with little wobble. In DMPC/DMPG, with a positive spontaneous curvature, BP100 at higher concentrations became tilted obliquely into the membrane, with the uncharged C-terminus inserted more deeply into the lipid bilayer, experiencing significant fluctuations in tilt angle. In DMPC/DMPG/lyso-MPC, with a pronounced positive spontaneous curvature, the helix tilted even further and became even more mobile. The 11-mer BP100 is obviously too short to form transmembrane pores. We conclude that BP100 operates via a carpet mechanism, whereby the C-terminus gets inserted into the hydrophobic core of the bilayer, which leads to membrane perturbation and induces transient permeability.
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http://dx.doi.org/10.1016/j.bbamem.2016.03.014DOI Listing
June 2016

Oriented Circular Dichroism: A Method to Characterize Membrane-Active Peptides in Oriented Lipid Bilayers.

Acc Chem Res 2016 Feb 12;49(2):184-92. Epub 2016 Jan 12.

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

The structures of membrane-bound polypeptides are intimately related to their functions and may change dramatically with the lipid environment. Circular dichroism (CD) is a rapid analytical method that requires relatively low amounts of material and no labeling. Conventional CD is routinely used to monitor the secondary structure of peptides and proteins in solution, for example, in the presence of ligands and other binding partners. In the case of membrane-active peptides and transmembrane proteins, these measurements can be applied to, and remain limited to, samples containing detergent micelles or small sonicated lipid vesicles. Such traditional CD analysis reveals only secondary structures. With the help of an oriented circular dichroism (OCD) setup, however, based on the preparation of macroscopically oriented lipid bilayers, it is possible to address the membrane alignment of a peptide in addition to its conformation. This approach has been mostly used for α-helical peptides so far, but other structural elements are conceivable as well. OCD analysis relies on Moffitt's theory, which predicts that the electronic transition dipole moments of the backbone amide bonds in helical polypeptides are polarized either parallel or perpendicular to the helix axis. The interaction of the electric field vector of the circularly polarized light with these transitions results in an OCD spectrum of a membrane-bound α-helical peptide, which exhibits a characteristic line shape and reflects the angle between the helix axis and the bilayer normal. For parallel alignment of a peptide helix with respect to the membrane surface (S-state), the corresponding "fingerprint" CD band around 208 nm will exhibit maximum negative amplitude. If the helix changes its alignment via an obliquely tilted (T-state) to a fully inserted transmembrane orientation (I-state), the ellipticity at 208 nm decreases and the value approaches zero due to the decreased interactions between the field and the transition dipole. Compared to conventional CD, OCD data are not only collected in the biologically relevant environment of a highly hydrated planar lipid bilayer (whose composition can be varied at will), but in addition it provides information about the tilt angle of the polypeptide in the membrane. It is the method of choice for screening numerous different conditions, such as peptide concentration, lipid composition, membrane additives, pH, temperature, and sample hydration. All these factors have been found to affect the peptide alignment in membrane, while having little or no influence on conformation. In many cases, the observed realignment could be related to biological action, such as pore formation by antimicrobial and cell-penetrating peptides, or to binding events of transmembrane segments of integral membrane proteins. Likewise, any lipid-induced conversion from α-helix to β-sheeted conformation is readily picked up by OCD and has been interpreted in terms of protein instability or amyloid-formation.
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http://dx.doi.org/10.1021/acs.accounts.5b00346DOI Listing
February 2016

Hydrophobic Mismatch Drives the Interaction of E5 with the Transmembrane Segment of PDGF Receptor.

Biophys J 2015 Aug;109(4):737-49

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology, Karlsruhe, Germany; Institute of Organic Chemistry, Karlsruhe Institute of Technology, Karlsruhe, Germany. Electronic address:

The oncogenic E5 protein from bovine papillomavirus is a short (44 amino acids long) integral membrane protein that forms homodimers. It activates platelet-derived growth factor receptor (PDGFR) β in a ligand-independent manner by transmembrane helix-helix interactions. The nature of this recognition event remains elusive, as numerous mutations are tolerated in the E5 transmembrane segment, with the exception of one hydrogen-bonding residue. Here, we examined the conformation, stability, and alignment of the E5 protein in fluid lipid membranes of substantially varying bilayer thickness, in both the absence and presence of the PDGFR transmembrane segment. Quantitative synchrotron radiation circular dichroism analysis revealed a very long transmembrane helix for E5 of ∼26 amino acids. Oriented circular dichroism and solid-state (15)N-NMR showed that the alignment and stability of this unusually long segment depend critically on the membrane thickness. When reconstituted alone in exceptionally thick DNPC lipid bilayers, the E5 helix was found to be inserted almost upright. In moderately thick bilayers (DErPC and DEiPC), it started to tilt and became slightly deformed, and finally it became aggregated in conventional DOPC, POPC, and DMPC membranes due to hydrophobic mismatch. On the other hand, when E5 was co-reconstituted with the transmembrane segment of PDGFR, it was able to tolerate even the most pronounced mismatch and was stabilized by binding to the receptor, which has the same hydrophobic length. As E5 is known to activate PDGFR within the thin membranes of the Golgi compartment, we suggest that the intrinsic hydrophobic mismatch of these two interaction partners drives them together. They seem to recognize each other by forming a closely packed bundle of mutually aligned transmembrane helices, which is further stabilized by a specific pair of hydrogen-bonding residues.
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http://dx.doi.org/10.1016/j.bpj.2015.07.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547410PMC
August 2015

UV-CD12: synchrotron radiation circular dichroism beamline at ANKA.

J Synchrotron Radiat 2015 May 11;22(3):844-52. Epub 2015 Apr 11.

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

Synchrotron radiation circular dichroism (SRCD) is a rapidly growing technique for structure analysis of proteins and other chiral biomaterials. UV-CD12 is a high-flux SRCD beamline installed at the ANKA synchrotron, to which it had been transferred after the closure of the SRS Daresbury. The beamline covers an extended vacuum-UV to near-UV spectral range and has been open for users since October 2011. The current end-station allows for temperature-controlled steady-state SRCD spectroscopy, including routine automated thermal scans of microlitre volumes of water-soluble proteins down to 170 nm. It offers an excellent signal-to-noise ratio over the whole accessible spectral range. The technique of oriented circular dichroism (OCD) was recently implemented for determining the membrane alignment of α-helical peptides and proteins in macroscopically oriented lipid bilayers as mimics of cellular membranes. It offers improved spectral quality <200 nm compared with an OCD setup adapted to a bench-top instrument, and accelerated data collection by a factor of ∼3. In addition, it permits investigations of low hydrated protein films down to 130 nm using a rotatable sample cell that avoids linear dichroism artifacts.
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http://dx.doi.org/10.1107/S1600577515004476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416691PMC
May 2015

Influence of hydrophobic residues on the activity of the antimicrobial peptide magainin 2 and its synergy with PGLa.

J Pept Sci 2015 May 22;21(5):436-45. Epub 2015 Apr 22.

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

Magainin 2 (MAG2) and PGLa are two related antimicrobial peptides found in the skin of the African frog Xenopus laevis with a pronounced synergistic activity, which act by permeabilizing bacterial membranes. To probe the influence of hydrophobic peptide-lipid and peptide-peptide interactions on the antimicrobial activity and on synergy, the sequence of MAG2 was modified by replacing single amino acids either with a small alanine or with the stiff and bulky hydrophobic 3-(trifluoromethyl)-L-bicyclopent-[1.1.1]-1-ylglycine side chain. The minimum inhibitory concentration of 14 MAG2 analogs was strongly influenced by these single substitutions: the antimicrobial activity was consistently improved when the hydrophobicity was increased on the hydrophobic face of the amphiphilic helix, while the activity decreased when the hydrophobicity was reduced. The synergy with PGLa, on the other hand, was rather insensitive to mutations of hydrophobic residues. It thus seems that the antimicrobial effect of MAG2 on its own depends strongly on the hydrophobicity of the peptide, while the synergy with PGLa does not depend on the overall hydrophobicity of MAG2.
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http://dx.doi.org/10.1002/psc.2780DOI Listing
May 2015

Hydrophobic mismatch demonstrated for membranolytic peptides, and their use as molecular rulers to measure bilayer thickness in native cells.

Sci Rep 2015 Mar 25;5:9388. Epub 2015 Mar 25.

1] Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), POB 3640, 76021 Karlsruhe, Germany [2] KIT, Institute of Organic Chemistry, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany.

Hydrophobic mismatch is a well-recognized principle in the interaction of transmembrane proteins with lipid bilayers. This concept was extended here to amphipathic membranolytic α-helices. Nine peptides with lengths between 14 and 28 amino acids were designed from repeated KIAGKIA motifs, and their helical nature was confirmed by circular dichroism spectroscopy. Biological assays for antimicrobial activity and hemolysis, as well as fluorescence vesicle leakage and solid-state NMR spectroscopy, were used to correlate peptide length with membranolytic activity. These data show that the formation of transmembrane pores is only possible under the condition of hydrophobic matching: the peptides have to be long enough to span the hydrophobic bilayer core to be able to induce vesicle leakage, kill bacteria, and cause hemolysis. By correlating the threshold lengths for biological activity with the biophysical results on model vesicles, the peptides could be utilized as molecular rulers to measure the membrane thickness in different cells.
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http://dx.doi.org/10.1038/srep09388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224518PMC
March 2015

Crown ether helical peptides are preferentially inserted in lipid bilayers as a transmembrane ion channels.

Biopolymers 2015 Jul;104(4):427-33

Faculté des Sciences et de Génie, Département de chimie and PROTEO, Université Laval, Québec, QC, G1V 0A6, Canada.

Oriented circular dichroism was used to study the alignment crown ether-modified peptides. The influence of different N- and C-functionalities was assessed using at variable peptide:lipid ratios from 1:20 to 1:200. Neither the functionalities nor the concentration had any major effect on the orientation. The alignment of the 21-mer peptides was also examined with lipid membranes of different bilayer thickness. The use of synchrotron radiation as light source allowed the study of peptide:lipid molar ratios from 1:20 to 1:1000. For all conditions studied, the peptides were found to be predominantly incorporated as a transmembrane helix into the membrane, especially at low peptide concentration, but started to aggregate on the membrane surface at higher peptide:lipid ratios. The structural information on the preferred trans-bilayer alignment of the crown ether functional groups explains their ion conductivity and is useful for the further development of membrane-active nanochemotherapeutics.
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http://dx.doi.org/10.1002/bip.22633DOI Listing
July 2015

Planar-chiral building blocks for metal-organic frameworks.

Chem Commun (Camb) 2015 Mar;51(23):4796-8

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

The first example of a planar-chiral building block being used for chiral metal-organic frameworks (MOFs) is presented. The porous MOF structure combined with the chiral properties of the planar linker allows a selective adsorption, demonstrated for a nonpolar terpene limonene in thin surface-mounted MOF films.
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http://dx.doi.org/10.1039/c5cc00694eDOI Listing
March 2015

Enhanced amphiphilic profile of a short β-stranded peptide improves its antimicrobial activity.

PLoS One 2015 24;10(1):e0116379. Epub 2015 Jan 24.

Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria, I-09042 Monserrato (CA), Italy.

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH2, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH2, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state 19F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β-SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which also leads to a dramatic alteration of membrane integrity as shown by 31P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic β-strands.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116379PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4305290PMC
February 2016

Structural characterization of a C-terminally truncated E5 oncoprotein from papillomavirus in lipid bilayers.

Biol Chem 2014 Dec;395(12):1443-52

E5 is the major transforming oncoprotein of bovine papillomavirus, which activates the platelet-derived growth factor receptor β in a highly specific manner. The short transmembrane protein E5 with only 44 residues interacts directly with the transmembrane segments of the receptor, but structural details are not available. Biophysical investigations are challenging, because the hydrophobic E5 protein tends to aggregate and get cross-linked non-specifically via two Cys residues near its C-terminus. Here, we demonstrate that a truncation by 10 amino acids creates a more manageable protein that can be conveniently used for structure analysis. Synchrotron radiation circular dichroism and solid-state (15)N- and (31)P-nuclear magnetic resonance spectroscopy show that this E5 variant serves as a representative model for the wild-type protein. The helical conformation of the transmembrane segment, its orientation in the lipid bilayer, and the ability to form homodimers in the membrane are not affected by the C-terminal truncation.
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http://dx.doi.org/10.1515/hsz-2014-0222DOI Listing
December 2014

Determination of the absolute configuration of perylene quinone-derived mycotoxins by measurement and calculation of electronic circular dichroism spectra and specific rotations.

Chemistry 2014 Sep 23;20(36):11463-70. Epub 2014 Jul 23.

Institute for Organic Chemistry, Karlsruhe Institute for Technology (KIT), Campus South, Fritz-Haber-Weg 6, 76131 Karlsruhe (Germany), Fax: (+49) 721-608-47652.

Altertoxins I-III, alterlosins I and II, alteichin (alterperylenol), stemphyltoxins I-IV, stemphyperylenol, stemphytriol, 7-epi-8-hydroxyaltertoxin I, and 6-epi-stemphytriol are mycotoxins derived from perylene quinone, for which the absolute configuration was not known. Electronic circular dichroism (ECD) spectra were calculated for these compounds and compared with measured spectra of altertoxins I-III, alteichin, and stemphyltoxin III and with reported Cotton effects. Specific rotations were calculated and compared with reported specific rotations. The absolute configuration of all the toxins, except for stemphyltoxin IV, could thus be determined. The validity of the assignment was high whenever reported ECD data were available for comparison, and the validity was lower when the assignment was based only on the comparison of calculated and reported specific rotations. ECD spectra are intrinsically different for toxins with a biphenyl substructure and for toxins derived from dihydroanthracene.
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http://dx.doi.org/10.1002/chem.201402567DOI Listing
September 2014

Structure-based engineering of a minimal porin reveals loop-independent channel closure.

Biochemistry 2014 Jul 15;53(29):4826-38. Epub 2014 Jul 15.

Department of Chemistry, Philipps-University Marburg , Hans-Meerwein-Straße, 35032 Marburg, Germany.

Porins, like outer membrane protein G (OmpG) of Escherichia coli, are ideal templates among ion channels for protein and chemical engineering because of their robustness and simple architecture. OmpG shows fast transitions between open and closed states, which were attributed to loop 6 (L6). As flickering limits single-channel-based applications, we pruned L6 by either 8 or 12 amino acids. While the open probabilities of both L6 variants resemble that of native OmpG, their gating frequencies were reduced by 63 and 81%, respectively. Using the 3.2 Å structure of the shorter L6 variant in the open state, we engineered a minimal porin (220 amino acids), where all remaining extramembranous loops were truncated. Unexpectedly, this minimized porin still exhibited gating, but it was 5-fold less frequent than in OmpG. The residual gating of the minimal pore is hence independent of L6 rearrangements and involves narrowing of the ion conductance pathway most probably driven by global stretching-flexing deformations of the membrane-embedded β-barrel.
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http://dx.doi.org/10.1021/bi500660qDOI Listing
July 2014