Anne S. Ulrich

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Name:

Organization: Institute of Biological Interfaces (IBG-2) , Germany

Department: DFG-Center for Functional Nanostructures (CFN)

Title: (PhD)

TOPICS

Organic Chemistry

Inorganic Chemistry

Metal Organic Frameworks

Chemicals
References

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

Co-reporter:Parvesh Wadhwani;Nico Heidenreich;Benjamin Podeyn;Jochen Bürck

Biomaterials Science (2013-Present) 2017 vol. 5(Issue 4) pp:817-827

Publication Date(Web):2017/03/28

DOI:10.1039/C7BM00069C

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.

Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR

Co-reporter:Stephan L. Grage, Marc-Antoine Sani, Olivier Cheneval, Sónia Troeira Henriques, ... Anne S. Ulrich

Biophysical Journal 2017 Volume 112, Issue 4(Volume 112, Issue 4) pp:

Publication Date(Web):28 February 2017

DOI:10.1016/j.bpj.2016.12.040

Cyclotides are ultra-stable cyclic disulfide-rich peptides from plants. Their biophysical effects and medically interesting activities are related to their membrane-binding properties, with particularly high affinity for phosphatidylethanolamine lipids. In this study we were interested in understanding the molecular details of cyclotide-membrane interactions, specifically with regard to the spatial orientation of the cyclotide kalata B1 from Oldenlandia affinis when embedded in a lipid bilayer. Our experimental approach was based on the use of solid-state 19F-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive 19F-NMR probe. Its rigid connection to the kalata B1 backbone scaffold, together with the well-defined structure of the cyclotide, allowed us to calculate the protein alignment in the membrane directly from the orientation-sensitive 19F-NMR signal. The hydrophobic and polar residues on the surface of kalata B1 form well-separated patches, endowing this cyclotide with a pronounced amphipathicity. The peptide orientation, as determined by NMR, showed that this amphipathic structure matches the polar/apolar interface of the lipid bilayer very well. A location in the amphiphilic headgroup region of the bilayer was supported by 15N-NMR of uniformly labeled protein, and confirmed using solid-state 31P- and 2H-NMR. 31P-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the 2H-NMR order parameter profile of the acyl chains suggest membrane thinning, as typically observed for amphiphilic peptides embedded near the polar/apolar bilayer interface. Furthermore, from the 19F-NMR analysis two important charged residues, E7 and R28, were found to be positioned equatorially. The observed location thus would be favorable for the postulated binding of E7 to phosphatidylethanolamine lipid headgroups. Furthermore, it may be speculated that this pair of side chains could promote oligomerization of kalata B1 through electrostatic intermolecular contacts via their complementary charges.

Flexibility vs rigidity of amphipathic peptide conjugates when interacting with lipid bilayers

Co-reporter:Oleg Babii, Sergii Afonin, Tim Schober, Igor V. Komarov, Anne S. Ulrich

Biochimica et Biophysica Acta (BBA) - Biomembranes 2017 Volume 1859, Issue 12(Issue 12) pp:

Publication Date(Web):1 December 2017

DOI:10.1016/j.bbamem.2017.09.021

•DAET building blocks can be used to study rigidity/flexibility effects in supramolecular model systems.•Photoswitchable DAET linkers perturb only up to 3–4 adjacent amino acid residues.•Membrane-bound amphiphilic secondary structure elements exert a negligible influence on each other when linked by DAET.•The rigidity of peptide conjugates affected their structural behavior only in the lipid gel phase.For the first time, the photoisomerization of a diarylethene moiety (DAET) in peptide conjugates was used to probe the effects of molecular rigidity/flexibility on the structure and behavior of model peptides bound to lipid membranes. The DAET unit was incorporated into the backbones of linear peptide-based constructs, connecting two amphipathic sequences (derived from the β-stranded peptide (KIGAKI)3 and/or the α-helical peptide BP100). A β-strand-DAET-α-helix and an α-helix-DAET-α-helix models were synthesized and studied in phospholipid membranes. Light-induced photoisomerization of the linker allowed the generation of two forms of each conjugate, which differed in the conformational mobility of the junction between the α-helical and/or the β-stranded part of these peptidomimetic molecules. A detailed study of their structural, orientational and conformational behavior, both in isotropic solution and in phospholipid model membranes, was carried out using circular dichroism and solid-state 19F-NMR spectroscopy. The study showed that the rigid and flexible forms of the two conjugates had appreciably different structures only when embedded in an anisotropic lipid environment and only in the gel phase. The influence of the rigidity/flexibility of the studied conjugates on the lipid thermotropic phase transition was also investigated by differential scanning calorimetry. Both models were found to destabilize the lamellar gel phases.Download high-res image (177KB)Download full-size image

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

Co-reporter:Philipp Mühlhäuser, Parvesh Wadhwani, Erik Strandberg, Jochen Bürck, Anne S. Ulrich

Biochimica et Biophysica Acta (BBA) - Biomembranes 2017 Volume 1859, Issue 12(Issue 12) pp:

Publication Date(Web):1 December 2017

DOI:10.1016/j.bbamem.2017.09.004

•SSL-25 was studied in lipid bilayers by oriented CD and solid-state 19F and 15N NMR.•The N-terminal half of SSL-25 forms an amphiphilic α-helix in the membrane-bound state.•The glycine-rich C-terminal half is disordered in the membrane-bound state.•The SSL-25 helix lies flat on the membrane surface, regardless of the lipid system.•The peptide binds to bacterial membranes but not to eukaryotic model membranes.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 19F-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 19F 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 19F 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.Download high-res image (137KB)Download full-size image

Diphytanoyl lipids as model systems for studying membrane-active peptides

Co-reporter:Sezgin Kara, Sergii Afonin, Oleg Babii, Anton N. Tkachenko, Igor V. Komarov, Anne S. Ulrich

Biochimica et Biophysica Acta (BBA) - Biomembranes 2017 Volume 1859, Issue 10(Issue 10) pp:

Publication Date(Web):1 October 2017

DOI:10.1016/j.bbamem.2017.06.003

•Oriented bilayers from diphytanoyl lipids are stable only in certain temperature/hydration ranges.•DPhPG, DPhPE, DPhPS form non-lamellar phases on solid supports under ambient conditions.•Mixtures of DPhPC with DPhPG can contain significant non-bilayer fractions.•PGLa does not insert into DPhPC membranes even at high peptide concentration.•In oriented circular dichroism analysis, an S-state can be misinterpreted when the formation of a bilayer is not confirmed.The branched chains in diphytanoyl lipids provide membranes with unique properties, such as high chemical/physical stability, low water permeability, and no gel-to-fluid phase transition at ambient temperature. Synthetic diphytanoyl phospholipids are often used as model membranes for electrophysiological experiments. To evaluate whether these sturdy lipids are also suitable for solid-state NMR, we have examined their interactions with a typical amphiphilic peptide in comparison with straight-chain lipids. First, their phase properties were monitored using 31P NMR, and the structural behaviour of the antimicrobial peptide PGLa was studied by 19F NMR and circular dichroism in oriented membrane samples. Only lipids with choline headgroups (DPhPC) were found to form stable lipid bilayers in oriented samples, while DPhPG, DPhPE and DPhPS display non-lamellar structures. Hence, the experimental temperature and hydration are crucial factors when using supported diphytanoyl lipids, as both parameters must be maintained in an appropriate range to avoid the formation of non-bilayer structures. For the same reason, a high content of other diphytanoyl lipids besides DPhPC in mixed lipid systems is not favourable. Unlike the situation in straight-chain membranes, we found that the α-helical PGLa was not able to insert into the tightly packed fluid bilayer of DPhPC but remained in a surface-bound state even at very high peptide concentration. This behaviour can be explained by the high cohesivity and the negative spontaneous curvature of the diphytanoyl lipids. These characteristic features must therefore be taken into consideration, both, in electrophysiological studies, and when interpreting the structural behaviour of membrane-active peptides in such lipid environment.Download high-res image (288KB)Download full-size image

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

Co-reporter:Jochen Bürck, Parvesh Wadhwani, Susanne Fanghänel, and Anne S. Ulrich

Accounts of Chemical Research 2016 Volume 49(Issue 2) pp:184

Publication Date(Web):January 12, 2016

DOI:10.1021/acs.accounts.5b00346

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.

Direct Photocontrol of Peptidomimetics: An Alternative to Oxygen-Dependent Photodynamic Cancer Therapy

Co-reporter:Dr. Oleg Babii;Dr. Sergii Afonin; Liudmyla V. Garmanchuk;Dr. Viktoria V. Nikulina;Tetiana V. Nikolaienko;Olha V. Storozhuk;Dmytro V. Shelest;Dr. Olga I. Dasyukevich; Liudmyla I. Ostapchenko;Dr. Volodymyr Iurchenko;Dr. Sergey Zozulya; Anne S. Ulrich; Igor V. Komarov

Angewandte Chemie International Edition 2016 Volume 55( Issue 18) pp:5493-5496

Publication Date(Web):

DOI:10.1002/anie.201600506

Abstract

Conventional photodynamic treatment strategies are based on the principle of activating molecular oxygen in situ by light, mediated by a photosensitizer, which leads to the generation of reactive oxygen species and thereby causes cell death. A diarylethene-derived peptidomimetic is presented that is suitable for photodynamic cancer therapy without any involvement of oxygen. This light-sensitive molecule is not a mediator but is itself the cytotoxic agent. As a derivative of the cyclic amphiphilic peptide gramicidin S, the peptidomimetic exists in two thermally stable photoforms that are interconvertible by light of different wavelengths. The isomer generated by visible light shows much stronger toxicity against tumor cells than the UV-generated isomer. First in vivo applications are demonstrated on a tumor animal model to illustrate how the peptidomimetic can be administered in the less toxic form and then activated locally in a solid tumor by visible light.

Direct Photocontrol of Peptidomimetics: An Alternative to Oxygen-Dependent Photodynamic Cancer Therapy

Angewandte Chemie 2016 Volume 128( Issue 18) pp:5583-5586

Publication Date(Web):

DOI:10.1002/ange.201600506

Abstract

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

Co-reporter:Jonathan Zerweck;Erik Strandberg;Jochen Bürck

European Biophysics Journal 2016 Volume 45( Issue 6) pp:535-547

Publication Date(Web):2016 September

DOI:10.1007/s00249-016-1120-7

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 15N-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 19F-NMR analysis.

Extending the Hydrophobic Mismatch Concept to Amphiphilic Membranolytic Peptides

Co-reporter:Ariadna Grau-Campistany; Erik Strandberg; Parvesh Wadhwani; Francesc Rabanal

The Journal of Physical Chemistry Letters 2016 Volume 7(Issue 7) pp:1116-1120

Publication Date(Web):March 10, 2016

DOI:10.1021/acs.jpclett.6b00136

A series of nine amphiphilic, pore-forming α-helical KIA peptides (KIAGKIA repeats) with lengths between 14 and 28 residues were studied by solid-state 15N NMR to determine their alignment in oriented lipid bilayers. In a 2:1 mixture of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) with its corresponding 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-MPC), which has a highly positive spontaneous curvature, the helix tilt angle was found to vary steadily with peptide length. The shortest peptide was aligned transmembrane and upright, while the longer ones successively became tilted away from the membrane normal. This behavior is in agreement with the hydrophobic matching concept, conceived so far only for hydrophobic helices. In 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, with a negative spontaneous curvature, all KIA peptides remained flat on the bilayer surface, while the cylindrical DMPC lipids permitted a slight tilt. Peptide insertion thus depends critically on the intrinsic lipid curvature, and helix orientation is then fine-tuned by membrane thickness. A refined toroidal pore model is proposed.

γ-(S)-Trifluoromethyl proline: evaluation as a structural substitute of proline for solid state 19F-NMR peptide studies

Co-reporter:Vladimir Kubyshkin, Sergii Afonin, Sezgin Kara, Nediljko Budisa, Pavel K. Mykhailiuk and Anne S. Ulrich

Organic & Biomolecular Chemistry 2015 vol. 13(Issue 11) pp:3171-3181

Publication Date(Web):13 Feb 2015

DOI:10.1039/C5OB00034C

γ-(4S)-Trifluoromethyl proline was synthesised according to a modified literature protocol with improved yield on a multigram scale. Conformational properties of the amide bond formed by the amino acid were characterised using N-acetyl methyl ester model. The amide populations (s-trans vs. s-cis) and thermodynamic parameters of the isomerization were found to be similar to the corresponding values for intact proline. Therefore, the γ-trifluoromethyl proline was suggested as a structurally low-disturbing proline substitution in peptides for their structural studies by 19F-NMR. Indeed, the exchange of native proline for γ-trifluoromethyl proline in the peptide antibiotic gramicidin S was shown to preserve the overall amphipathic peptide structure. The utility of the amino acid as a selective 19F-NMR label was demonstrated by observing the re-alignment of the labelled gramicidin S in oriented lipid bilayers.

Design and Synthesis of a Monofluoro-Substituted Aromatic Amino Acid as a Conformationally Restricted 19F NMR Label for Membrane-Bound Peptides

Co-reporter:Anton N. Tkachenko;Pavel K. Mykhailiuk;Dmytro S. Radchenko;Oleg Babii;Sergii Afonin;Igor V. Komarov

European Journal of Organic Chemistry 2014 Volume 2014( Issue 17) pp:3584-3591

Publication Date(Web):

DOI:10.1002/ejoc.201301737

Abstract

A monofluoro-substituted amino acid was designed to serve as a conformationally restricted label for solid-state ¹⁹F NMR distance measurements in membrane-bound peptides. The aromatic cis and trans isomers of 1-amino-3-(4-fluorophenyl)cyclobutanecarboxylic acid were synthesized in five steps from diethyl 2-(4-fluorophenyl)propanedioate. They were incorporated into the antimicrobial peptide gramicidin S to replace a native ^DPhenylalanine residue. Because the Cα-tetrasubstituted amino acid cannot racemize, it showed full compatibility with solid-phase peptide synthesis protocols. According to circular dichroism analysis and molecular modeling, the ¹⁹F-labeled analogues of the known helix-inducing amino acid (1-aminocyclobutane-1-carboxylic acid) do not disrupt the peptide conformation when substituted for Phe, neither in a β-turn nor in an α-helix.

19F-Labeling of Peptides Revealing Long-Range NMR Distances in Fluid Membranes

Co-reporter:Stephan L. Grage, Xiaojun Xu, Markus Schmitt, Parvesh Wadhwani, and Anne S. Ulrich

The Journal of Physical Chemistry Letters 2014 Volume 5(Issue 24) pp:4256-4259

Publication Date(Web):November 19, 2014

DOI:10.1021/jz502195t

NMR distance measurements lie at the heart of structural biology. However, long-range distances could not yet be detected in liquid–crystalline biomembranes, because dipolar couplings are partially averaged by the intrinsic molecular mobility. Using conformationally constrained 19F-labeled amino acids as reporter groups, we could more than double the accessible interatomic distance range by combining a highly sensitive solid-state multipulse 19F-NMR scheme with a favorable sample geometry. Two rigid 4F-phenylglycine labels were placed into the helical antimicrobial peptide PGLa embedded in fluid oriented membrane samples. A modified Carr–Purcell–Meiboom–Gill sequence yielded an intramolecular distance of 6.6 Å for the labels spanning one helix turn, and 11.0 Å was obtained when the labels spanned two turns. This approach should now also allow the characterization of conformational changes in membrane-active peptides and of oligomeric assemblies in a biologically relevant lipid environment.Keywords: conformationally constrained amino acids; liquid−crystalline phase biomembranes; long-range interatomic distance; membrane-active peptides; monofluorine substituents; solid-state 19F-NMR;

Innenrcktitelbild: Controlling Biological Activity with Light: Diarylethene-Containing Cyclic Peptidomimetics (Angew. Chem. 13/2014)

Co-reporter:Oleg Babii;Dr. Sergii Afonin;Dr. Marina Berditsch;Sabine Reiβer;Dr. Pavel K. Mykhailiuk;Dr. Vladimir S. Kubyshkin;Dr. Thomas Steinbrecher; Anne S. Ulrich; Igor V. Komarov

Angewandte Chemie 2014 Volume 126( Issue 13) pp:

Publication Date(Web):

DOI:10.1002/ange.201401081

Controlling Biological Activity with Light: Diarylethene-Containing Cyclic Peptidomimetics

Co-reporter:Oleg Babii;Dr. Sergii Afonin;Dr. Marina Berditsch;Sabine Reiβer;Dr. Pavel K. Mykhailiuk;Dr. Vladimir S. Kubyshkin;Dr. Thomas Steinbrecher; Anne S. Ulrich; Igor V. Komarov

Angewandte Chemie 2014 Volume 126( Issue 13) pp:3460-3463

Publication Date(Web):

DOI:10.1002/ange.201310019

Abstract

Photobiological processes in nature are usually triggered by nonpeptidic chromophores or by modified side chains. A system is presented in which the polypeptide backbone itself can be conformationally switched by light. An amino acid analogue was designed and synthesized based on a reversibly photoisomerizable diarylethene scaffold. This analogue was incorporated into the cyclic backbone of the antimicrobial peptide gramicidin S at several sites. The biological activity of the resulting peptidomimetics could then be effectively controlled by ultraviolet/visible light within strictly defined spatial and temporal limits.

Controlling Biological Activity with Light: Diarylethene-Containing Cyclic Peptidomimetics

Co-reporter:Oleg Babii;Dr. Sergii Afonin;Dr. Marina Berditsch;Sabine Reiβer;Dr. Pavel K. Mykhailiuk;Dr. Vladimir S. Kubyshkin;Dr. Thomas Steinbrecher; Anne S. Ulrich; Igor V. Komarov

Angewandte Chemie International Edition 2014 Volume 53( Issue 13) pp:3392-3395

Publication Date(Web):

DOI:10.1002/anie.201310019

Abstract

Inside Back Cover: Controlling Biological Activity with Light: Diarylethene-Containing Cyclic Peptidomimetics (Angew. Chem. Int. Ed. 13/2014)

Co-reporter:Oleg Babii;Dr. Sergii Afonin;Dr. Marina Berditsch;Sabine Reiβer;Dr. Pavel K. Mykhailiuk;Dr. Vladimir S. Kubyshkin;Dr. Thomas Steinbrecher; Anne S. Ulrich; Igor V. Komarov

Angewandte Chemie International Edition 2014 Volume 53( Issue 13) pp:

Publication Date(Web):

DOI:10.1002/anie.201401081

Oriented Circular Dichroism Analysis of Chiral Surface-Anchored MetalOrganic Frameworks Grown by Liquid-Phase Epitaxy and upon Loading with Chiral Guest Compounds

Co-reporter:Zhi-Gang Gu;Dr. Jochen Bürck;Dr. Angela Bihlmeier;Dr. Jinxuan Liu;Dr. Osama Shekhah;Dr. Peter G. Weidler;Dr. Carlos Azucena;Zhengbang Wang;Stefan Heissler;Dr. Hartmut Gliemann;Dr. Wim Klopper;Dr. Anne S. Ulrich;Dr. Christof Wöll

Chemistry - A European Journal 2014 Volume 20( Issue 32) pp:9879-9882

Publication Date(Web):

DOI:10.1002/chem.201403524

Abstract

Oriented circular dichroism (OCD) is explored and successfully applied to investigate chiral surface-anchored metal–organic frameworks (SURMOFs) based on camphoric acid (D- and Lcam) with the composition [Cu₂(Dcam)_2x(Lcam)_2−2x(dabco)]_n (dabco=1,4-diazabicyclo-[2.2.2]-octane). The three-dimensional chiral SURMOFs with high-quality orientation were grown on quartz glass plates by using a layer-by-layer liquid-phase epitaxy method. The growth orientation, as determined by X-ray diffraction (XRD), could be switched between the [001] and [110] direction by using either OH- or COOH-terminated substrates. These SURMOFs were characterized by using OCD, which confirmed the ratio as well as the orientation of the enantiomeric linker molecules. Theoretical computations demonstrate that the OCD band intensities of the enantiopure [Cu₂(Dcam)₂(dabco)]_n grown in different orientations are a direct result of the anisotropic nature of the chiral SURMOFs. Finally, the enantiopure [Cu₂(Dcam)₂(dabco)]_n and [Cu₂(Lcam)₂(dabco)]_n SURMOFs were loaded with the two chiral forms of ethyl lactate [(+)-ethyl-D-lactate and (−)-ethyl-L-lactate)]. An enantioselective enrichment of >60 % was observed by OCD when the chiral host scaffold was loaded from the racemic mixture.

Stereochemical effects on the aggregation and biological properties of the fibril-forming peptide [KIGAKI]3 in membranes

Co-reporter:Parvesh Wadhwani, Johannes Reichert, Erik Strandberg, Jochen Bürck, Julia Misiewicz, Sergii Afonin, Nico Heidenreich, Susanne Fanghänel, Pavel K. Mykhailiuk, Igor V. Komarov and Anne S. Ulrich

Physical Chemistry Chemical Physics 2013 vol. 15(Issue 23) pp:8962-8971

Publication Date(Web):07 May 2013

DOI:10.1039/C3CP50896J

Single D-amino acid substitutions can be used to suppress or slow down the aggregation of peptides into β-sheeted assemblies compared to the respective L-amino acids. Here, we investigate the influence of local stereochemistry in the model peptide [KIGAKI]3-NH2, which is known to form amyloid-like fibrils. To find out whether aggregation plays a role in various biologically relevant functions that involve peptide–lipid interactions, we studied the antimicrobial, hemolytic and fusogenic activities of this amphiphilic membrane-active molecule. The stiff and sterically constrained amino acid CF3-Bpg [3-(trifluoromethyl)-bicyclopent-[1,1,1]-1-ylglycine] was incorporated either as an L- or a D-enantiomer at different hydrophobic positions of the KIGAKI sequence. D-Epimers have a higher aggregation threshold than the L-epimers, yet the aggregation of both was confirmed using electron microscopy and circular dichroism. Solid-state 19F-NMR analysis showed that the peptide aggregated in native membranes from human erythrocytes and bacterial protoplasts in the same way as in synthetic lipid bilayers. We then monitored the effect of the single L- or D-CF3-Bpg substitutions in KIGAKI on its distinct biological activities, which have to be measured at low peptide concentrations where the aggregation threshold cannot be directly assessed. These functional assays showed that the aggregation propensity of KIGAKI does not play a role in its antimicrobial action, but an increased tendency to aggregate promotes other undesirable effects such as hemolysis and membrane fusion. These results confirm the membranolytic and thereby toxic nature of amyloidogenic peptides, and emphasize the unpredictable role of peptide aggregation in the different assays used to study biological activities.

Incorporation of labile trans-4,5-difluoromethanoproline into a peptide as a stable label for 19F NMR structure analysis

Co-reporter:Vladimir S. Kubyshkin, Pavel K. Mykhailiuk, Sergii Afonin, Stephan L. Grage, Igor V. Komarov, Anne S. Ulrich

Journal of Fluorine Chemistry 2013 Volume 152() pp:136-143

Publication Date(Web):August 2013

DOI:10.1016/j.jfluchem.2013.03.002

•Intrinsically unstable amino acid was incorporated into a cyclic peptide (gramicidin S).•Difluorocyclopropanation was done on the stable dipeptide building block.•The amino acid did not perturb the conformation and function of gramicidin S.•The peptide was evaluated in solid state 19F NMR structure analysis.•Geminal-F2 system delivers several orientational constrain at one spectrum.Trans-4,5-Difluoromethano-proline was incorporated into the cyclic antimicrobial peptide gramicidin S in place of a native proline residue. Introduction of this intrinsically unstable amino acid into the polypeptide backbone was achieved using a dipeptide strategy. The stable dipeptide building block with the N-acylated 4,5-difluoromethano-proline fragment was obtained by direct difluorocyclopropanation of an unsaturated precursor. The influence of the unnatural amino acid on the conformation and function of gramicidin S was evaluated using circular dichroism and biological assays. The application of trans-4,5-difluoromethano-proline as a new label for solid state 19F NMR structure analysis of membrane-active peptides was tested on gramicidin S and compared with previous labeling schemes.

A 19FNMR Label to Substitute Polar Amino Acids in Peptides: A CF3-Substituted Analogue of Serine and Threonine

Co-reporter:Anton N. Tkachenko;Dr. Pavel K. Mykhailiuk;Dr. Sergii Afonin;Dr. Dmytro S. Radchenko;Vladimir S. Kubyshkin; Anne S. Ulrich; Igor V. Komarov

Angewandte Chemie International Edition 2013 Volume 52( Issue 5) pp:1486-1489

Publication Date(Web):

DOI:10.1002/anie.201208069

Self-Assembly of Flexible β-Strands into Immobile Amyloid-Like β-Sheets in Membranes As Revealed by Solid-State 19F NMR

Co-reporter:Parvesh Wadhwani ; Erik Strandberg ; Nico Heidenreich ; Jochen Bürck ; Susanne Fanghänel

Journal of the American Chemical Society 2012 Volume 134(Issue 15) pp:6512-6515

Publication Date(Web):March 27, 2012

DOI:10.1021/ja301328f

The cationic peptide [KIGAKI]3 was designed as an amphiphilic β-strand and serves as a model for β-sheet aggregation in membranes. Here, we have characterized its molecular conformation, membrane alignment, and dynamic behavior using solid-state 19F NMR. A detailed structure analysis of selectively 19F-labeled peptides was carried out in oriented DMPC bilayers. It showed a concentration-dependent transition from monomeric β-strands to oligomeric β-sheets. In both states, the rigid 19F-labeled side chains project straight into the lipid bilayer but they experience very different mobilities. At low peptide-to-lipid ratios ≤1:400, monomeric [KIGAKI]3 swims around freely on the membrane surface and undergoes considerable motional averaging, with essentially uncoupled ϕ/ψ torsion angles. The flexibility of the peptide backbone in this 2D plane is reminiscent of intrinsically unstructured proteins in 3D. At high concentrations, [KIGAKI]3 self-assembles into immobilized β-sheets, which are untwisted and lie flat on the membrane surface as amyloid-like fibrils. This is the first time the transition of monomeric β-strands into oligomeric β-sheets has been characterized by solid-state NMR in lipid bilayers. It promises to be a valuable approach for studying membrane-induced amyloid formation of many other, clinically relevant peptide systems.

Alignment of Druglike Compounds in Lipid Bilayers Analyzed by Solid-State 19F-NMR and Molecular Dynamics, Based on Dipolar Couplings of Adjacent CF3 Groups

Co-reporter:Ulrich H. N. Dürr, Sergii Afonin, Barbara Hoff, Giuseppina de Luca, James W. Emsley, and Anne S. Ulrich

The Journal of Physical Chemistry B 2012 Volume 116(Issue 16) pp:4769-4782

Publication Date(Web):March 22, 2012

DOI:10.1021/jp212339k

Solid-state 19F-NMR spectroscopy is frequently used to analyze the structure and dynamics of lipophilic drugs and peptides embedded in biomembranes. The homonuclear dipolar couplings of trifluoromethyl (CF3) labels can provide valuable parameters such as orientational constraints and/or distances. To characterize the complex dipolar patterns of multiple 19F spin interactions, three different model compounds carrying two CF3 groups in meta-position on a phenyl ring were incorporated in macroscopically aligned DMPC bilayers. The dipolar patterns obtained with the CPMG (Carr–Purcell–Meiboom–Gill) multipulse sequence were analyzed to yield simultaneously the intra-CF3 and intergroup dipolar coupling values. The fluorine–fluorine distances were predicted by a density functional calculation, and the alignment of the labeled molecular segment could be determined from these distances and the dipolar coupling values. The different compounds were found to align in the lipid bilayer according to their amphiphilic properties, though with a weak anisotropic preference that is typical of solutes in liquid crystals. The residual dipolar couplings were used to calculate Saupe order parameters. For the least complex molecule, (CF3)2-BA, an orientational probability function for the solute in the lipid matrix could be derived. The overall description of how (CF3)2-BA is embedded in the bilayer was independently assessed by molecular dynamics simulations, and compared in structural and dynamical terms with the results of the NMR experiments.

Comparative analysis of the orientation of transmembrane peptides using solid-state 2H- and 15N-NMR: mobility matters

Co-reporter:Stephan L. Grage;Erik Strandberg;Parvesh Wadhwani

European Biophysics Journal 2012 Volume 41( Issue 5) pp:475-482

Publication Date(Web):2012 May

DOI:10.1007/s00249-012-0801-0

Many solid-state nuclear magnetic resonance (NMR) approaches for membrane proteins rely on orientation-dependent parameters, from which the alignment of peptide segments in the lipid bilayer can be calculated. Molecules embedded in liquid-crystalline membranes, such as monomeric helices, are highly mobile, leading to partial averaging of the measured NMR parameters. These dynamic effects need to be taken into account to avoid misinterpretation of NMR data. Here, we compare two common NMR approaches: 2H-NMR quadrupolar waves, and separated local field 15N–1H polarization inversion spin exchange at magic angle (PISEMA) spectra, in order to identify their strengths and drawbacks for correctly determining the orientation and mobility of α-helical transmembrane peptides. We first analyzed the model peptide WLP23 in oriented dimyristoylphosphatidylcholine (DMPC) membranes and then contrasted it with published data on GWALP23 in dilauroylphosphatidylcholine (DLPC). We only obtained consistent tilt angles from the two methods when taking dynamics into account. Interestingly, the two related peptides differ fundamentally in their mobility. Although both helices adopt the same tilt in their respective bilayers (~20°), WLP23 undergoes extensive fluctuations in its azimuthal rotation angle, whereas GWALP23 is much less dynamic. Both alternative NMR methods are suitable for characterizing orientation and dynamics, yet they can be optimally used to address different aspects. PISEMA spectra immediately reveal the presence of large-amplitude rotational fluctuations, which are not directly seen by 2H-NMR. On the other hand, PISEMA was unable to define the azimuthal rotation angle in the case of the highly dynamic WLP23, though the helix tilt could still be determined, irrespective of any dynamics parameters.

Antimicrobial and cell-penetrating peptides induce lipid vesicle fusion by folding and aggregation

Co-reporter:Parvesh Wadhwani;Johannes Reichert;Jochen Bürck

European Biophysics Journal 2012 Volume 41( Issue 2) pp:177-187

Publication Date(Web):2012 February

DOI:10.1007/s00249-011-0771-7

According to their distinct biological functions, membrane-active peptides are generally classified as antimicrobial (AMP), cell-penetrating (CPP), or fusion peptides (FP). The former two classes are known to have some structural and physicochemical similarities, but fusogenic peptides tend to have rather different features and sequences. Nevertheless, we found that many CPPs and some AMPs exhibit a pronounced fusogenic activity, as measured by a lipid mixing assay with vesicles composed of typical eukaryotic lipids. Compared to the HIV fusion peptide (FP23) as a representative standard, all designer-made peptides showed much higher lipid-mixing activities (MSI-103, MAP, transportan, penetratin, Pep1). Native sequences, on the other hand, were less fusogenic (magainin 2, PGLa, gramicidin S), and pre-aggregated ones were inactive (alamethicin, SAP). The peptide structures were characterized by circular dichroism before and after interacting with the lipid vesicles. A striking correlation between the extent of conformational change and the respective fusion activities was found for the series of peptides investigated here. At the same time, the CD data show that lipid mixing can be triggered by any type of conformation acquired upon binding, whether α-helical, β-stranded, or other. These observations suggest that lipid vesicle fusion can simply be driven by the energy released upon membrane binding, peptide folding, and possibly further aggregation. This comparative study of AMPs, CPPs, and FPs emphasizes the multifunctional aspects of membrane-active peptides, and it suggests that the origin of a peptide (native sequence or designer-made) may be more relevant to define its functional range than any given name.

A kinked antimicrobial peptide from Bombina maxima. II. Behavior in phospholipid bilayers

Co-reporter:Ralf Heinzmann;Stephan L. Grage;Constantin Schalck

European Biophysics Journal 2011 Volume 40( Issue 4) pp:463-470

Publication Date(Web):2011 April

DOI:10.1007/s00249-010-0668-x

The preceding contribution by Toke et al. has studied the structure of the cationic antimicrobial peptide maximin-4 in detergent micelles and in organic solvent, revealing a different kink angle and side-chain interactions in the two different environments. Here, we have examined the same peptide in lipid bilayers using oriented circular dichroism (OCD) and solid-state 15N nuclear magnetic resonance (NMR) in aligned samples. OCD showed that maximin-4 is helical and adopts an oblique alignment in the membrane, and lacks the characteristic realignment response that is often observed for amphipathic α-helical peptides at a peptide:lipid ratio between 1:100 and 1:20. Solid-state 15N-NMR experiments suggest that maximin-4 also remains unaffected by lipid charge and temperature. Analyzing 15N labels in positions Ala12, Ala13, and Leu14, an oblique tilt angle of the N-terminal helix of ~130° relative to the membrane normal was found, in good agreement with the amphiphilic profile of this segment. An additional constraint at Ala22 in the C-terminal segment is found to be compatible with a continuous α-helix, but unfavorable side-chain interactions make this solution unlikely. Instead, a kink at Gly16 seems fully compatible with all known constraints and with the biophysical expectations in the membrane-bound state, given the liquid-state NMR structures. It thus seems that the flexible kink in maximin-4 allows the two helical segments to adjust to the local environment. The irregular amphiphilic profile and the resulting versatility in shape might explain why maximin-4 lacks the realignment response that has been characteristically observed for many related frog peptides forming straight amphipathic α-helices.

Irregular structure of the HIV fusion peptide in membranes demonstrated by solid-state NMR and MD simulations

Co-reporter:Dorit Grasnick;Ulrich Sternberg;Erik Strandberg

European Biophysics Journal 2011 Volume 40( Issue 4) pp:529-543

Publication Date(Web):2011 April

DOI:10.1007/s00249-011-0676-5

To better understand peptide-induced membrane fusion at a molecular level, we set out to determine the structure of the fusogenic peptide FP23 from the HIV-1 protein gp41 when bound to a lipid bilayer. An established solid-state 19F nuclear magnetic resonance (NMR) approach was used to collect local orientational constraints from a series of CF3-phenylglycine-labeled peptide analogues in macroscopically aligned membranes. Fusion assays showed that these 19F-labels did not significantly affect peptide function. The NMR spectra were characteristic of well-behaved samples, without any signs of heterogeneity or peptide aggregation at 1:300 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). We can conclude from these NMR data that FP23 has a well-defined (time-averaged) conformation and undergoes lateral diffusion in the bilayer plane, presumably as a monomer or small oligomer. Attempts to evaluate its conformation in terms of various secondary structures, however, showed that FP23 does not form any type of regular helix or β-strand. Therefore, all-atom molecular dynamics (MD) simulations were carried out using the orientational NMR constraints as pseudo-forces to drive the peptide into a stable alignment and structure. The resulting picture suggests that FP23 can adopt multiple β-turns and insert obliquely into the membrane. Such irregular conformation explains why the structure of the fusion peptide could not be reliably determined by any biophysical method so far.

Special issue on membrane-active peptides

Co-reporter:Sergiy Afonin;Davor Juretić;Frances Separovic

European Biophysics Journal 2011 Volume 40( Issue 4) pp:347-348

Publication Date(Web):2011 April

DOI:10.1007/s00249-011-0670-y

19F NMR Analysis of the Antimicrobial Peptide PGLa Bound to Native Cell Membranes from Bacterial Protoplasts and Human Erythrocytes

Co-reporter:Marco Ieronimo ; Sergii Afonin ; Katja Koch ; Marina Berditsch ; Parvesh Wadhwani

Journal of the American Chemical Society 2010 Volume 132(Issue 26) pp:8822-8824

Publication Date(Web):June 15, 2010

DOI:10.1021/ja101608z

19F NMR is a unique tool to examine the structure of fluorine-labeled peptides in their native cellular environment, due to an exquisite sensitivity and lack of natural abundance background. For solid-state NMR analysis, we isolated native membranes from erythrocyte ghosts and bacterial protoplasts and prepared them as macroscopically oriented samples. They showed a high purity and quality of alignment according to 31P NMR, and the membrane-bound antimicrobial peptide PGLa could be detected by 19F NMR. The characteristic fingerprint splitting of its 19F reporter group indicated that the peptide helix binds to the native membranes in a surface alignment, albeit with a higher affinity in the prokaryotic than the eukaryotic system.

Membrane Alignment of the Pore-Forming Component TatAd of the Twin-Arginine Translocase from Bacillus subtilis Resolved by Solid-State NMR Spectroscopy

Co-reporter:Torsten H. Walther ; Stephan L. Grage ; Nadine Roth

Journal of the American Chemical Society 2010 Volume 132(Issue 45) pp:15945-15956

Publication Date(Web):October 26, 2010

DOI:10.1021/ja106963s

The twin-arginine translocase (Tat) provides protein export in bacteria and plant chloroplasts and is capable of transporting fully folded proteins across the membrane. We resolved the conformation and membrane alignment of the pore-forming subunit TatAd from Bacillus subtilis using solid-state NMR spectroscopy. The relevant structured part of the protein, TatA2−45, contains a transmembrane segment (TMS) and an amphiphilic helix (APH). It was reconstituted in planar bicelles, which represent the lipid environment of a bacterial membrane. The SAMMY solid-state NMR experiment was used to correlate 15N chemical shifts and 1H−15N dipolar couplings in the backbone and side chains of the 15N-labeled protein. The observed wheel-like patterns (“PISA wheels”) in the resulting 2-dimensional spectra confirm the α-helical character of the two segments and reveal their alignment in the lipid bilayer. Helix tilt angles (τTMS = 13°, τAPH = 64°) were obtained from uniformly labeled protein, and azimuthal rotations (ρVal15 = 235°, ρIle29 = 25°) were obtained from selective labels. These constraints define two distinct families of allowed structures for TatA in the membrane-bound state. The manifold of solutions could be narrowed down to a unique structure by using input from a liquid-state NMR study of TatA in detergent micelles, as recently described [Hu, Y.; Zhao, E.; Li, H.; Xia, B.; Jin, C. J. Am. Chem. Soc. 2010, DOI: 10.1021/ja1053785]. Interestingly, the APH showed an unexpectedly slanted alignment in the protein, different from that of the isolated APH peptide. This finding implies that the amphiphilic region of TatA is not just a flexible attachment to the transmembrane anchor but might be able to form intra- or even intermolecular salt-bridges, which could play a key role in pore assembly.

Chemical Labeling Strategy with (R)- and (S)-Trifluoromethylalanine for Solid State 19F NMR Analysis of Peptaibols in Membranes

Co-reporter:Daniel Maisch ; Parvesh Wadhwani ; Sergii Afonin ; Christoph Böttcher ; Beate Koksch

Journal of the American Chemical Society 2009 Volume 131(Issue 43) pp:15596-15597

Publication Date(Web):October 14, 2009

DOI:10.1021/ja9067595

Substitution of a single Aib-residue in a peptaibol with (R)- and (S)-trifluoromethylalanine yields two local orientational constraints θ by solid state 19F NMR. The structure of the membrane-perturbing antibiotic alamethicin in DMPC bilayers was analyzed in terms of two angles τ and ρ from six such constraints, showing that the N-terminus (up to a kink at Pro14) is folded as an α-helix, tilted away from the membrane normal by 8°, and assembled as an oligomer. The new 19F NMR label CF3-Ala has thus been demonstrated to be highly sensitive, virtually unperturbing, and ideally suited to characterize peptaibols in membranes.

Calculation of fluorine chemical shift tensors for the interpretation of oriented 19F-NMR spectra of gramicidin A in membranes

Co-reporter:Ulrich Sternberg, Marco Klipfel, Stephan L. Grage, Raiker Witter and Anne S. Ulrich

Physical Chemistry Chemical Physics 2009 vol. 11(Issue 32) pp:7048-7060

Publication Date(Web):20 Jul 2009

DOI:10.1039/B908236K

A semi-empirical method for the prediction of chemical shifts, based on bond polarization theory, has recently been introduced for 13C. Here, we extended this approach to calculate the 19F chemical shift tensors of fluorine bound to aromatic rings and in aliphatic CF3groups. For the necessary parametrization, ab initio chemical shift calculations were performed at the MP2 level for a set of fluorinated molecules including tryptophan. The bond polarization parameters obtained were used to calculate the 19F chemical shift tensors for several crystalline molecules, and to reference the calculated values on a chemical shift scale relative to CFCl3. As a first biophysical application, we examined the distribution of conformations of a 19F-labeled tryptophan side chain in the membrane-bound ion channel peptide, gramicidin A. The fluorine chemical shift tensors were calculated from snapshots of a molecular dynamics simulation employing the 19F-parametrized bond polarization theory. In this MD simulation, published 2H quadrupolar and 15N–1H dipolar couplings of the indole ring were used as orientational constraints to determine the conformational distribution of the 5F-Trp13 side chain. These conformations were then used to interpret the spectra of 19F-labeled gramicidin A in fluid and gel phase lipid bilayers.

Structure analysis of the membrane protein TatCd from the Tat system of B. subtilis by circular dichroism

Co-reporter:Olga V. Nolandt, Torsten H. Walther, Siegmar Roth, Jochen Bürck, Anne S. Ulrich

Biochimica et Biophysica Acta (BBA) - Biomembranes (October 2009) Volume 1788(Issue 10) pp:

Publication Date(Web):October 2009

DOI:10.1016/j.bbamem.2009.07.003

The twin arginine translocation (Tat) system can transport fully folded proteins, including their cofactors, across bacterial and thylakoid membranes. The Tat system of Bacillus subtilis that serves to export the phosphodiesterase (PhoD) consists of only two membrane proteins, TatAd and TatCd. The larger component TatCd has a molecular weight of 28 kDa and several membrane-spanning segments. This protein has been expressed in Escherichia coli and purified in sufficient amounts for structure analysis by circular dichroism (CD) and NMR spectroscopy. TatCd was reconstituted in detergent micelles and in lipid bilayers for CD analysis in solution and in macroscopically oriented samples, to examine the stability of the protein. Suitable protocols and model membrane systems have been established, by which TatCd maintains the level of helicity close to theoretically predicted, and its transmembrane alignment could been verified.

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

Co-reporter:Héctor Zamora-Carreras, Erik Strandberg, Philipp Mühlhäuser, Jochen Bürck, Parvesh Wadhwani, M. Ángeles Jiménez, Marta Bruix, Anne S. Ulrich

Biochimica et Biophysica Acta (BBA) - Biomembranes (June 2016) Volume 1858(Issue 6) pp:

Publication Date(Web):June 2016

DOI:10.1016/j.bbamem.2016.03.014

•BP100 is a short, multi-functional, membrane-active peptide.•An Ala-scan was performed and improved therapeutic peptides identified.•The peptide is highly helical in membranes.•The orientation in the membrane could be determined from solid-state 2H NMR.•BP100 membrane function is most likely based on a carpet mechanism.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 2H 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.Figure optionsDownload full-size imageDownload high-quality image (277 K)Download as PowerPoint slide

Synergistic transmembrane insertion of the heterodimeric PGLa/magainin 2 complex studied by solid-state NMR

Co-reporter:Erik Strandberg, Pierre Tremouilhac, Parvesh Wadhwani, Anne S. Ulrich

Biochimica et Biophysica Acta (BBA) - Biomembranes (August 2009) Volume 1788(Issue 8) pp:

Publication Date(Web):August 2009

DOI:10.1016/j.bbamem.2008.12.018

The skin secretions of amphibians are a rich source of antimicrobial peptides. The two antimicrobial peptides PGLa and magainin 2, isolated from the African frog Xenopus laevis, have been shown to act synergistically by permeabilizing the membranes of microorganisms. In this report, the literature on PGLa is extensively reviewed, with special focus on its synergistically enhanced activity in the presence of magainin 2. Our recent solid state 2H NMR studies of the orientation of PGLa in lipid membranes alone and in the presence of magainin 2 are described in detail, and some new data from 3,3,3-2H3-L-alanine labeled PGLa are included in the analysis.

Peptide-Lipid Interactions of the Stress-Response Peptide TisB That Induces Bacterial Persistence

Co-reporter:Thomas Steinbrecher, Sebastian Prock, Johannes Reichert, Parvesh Wadhwani, Benjamin Zimpfer, Jochen Bürck, Marina Berditsch, Marcus Elstner, Anne S. Ulrich

Biophysical Journal (3 October 2012) Volume 103(Issue 7) pp:

Publication Date(Web):3 October 2012

DOI:10.1016/j.bpj.2012.07.060

The bacterial stress-response peptide TisB in Escherichia coli has been suggested to dissipate the transmembrane potential, such that the depletion of ATP levels induces the formation of dormant persister cells which can eventually form biofilms. We studied the structure and membrane interactions of TisB to find out whether it forms pores or other proton-selective channels. Circular dichroism revealed an amphiphilic α-helical structure when reconstituted in lipid vesicles, and oriented circular dichroism showed that the helix assumes a transmembrane alignment. The addition of TisB to dye-loaded vesicles caused leakage only at very high peptide concentration, notably with a Hill coefficient of 2, which suggests that dimers must be involved. Coarse-grained molecular dynamics simulations showed that membrane binding of monomeric TisB is rapid and spontaneous, and transmembrane insertion is energetically feasible. When TisB oligomers are assembled as transmembrane pores, these channels collapse during the simulations, but transmembrane dimers are found to be stable. Given the pattern of charges on the amphiphilic TisB helix, we postulate that antiparallel dimers could be assembled via a ladder of salt bridges. This electrostatic charge-zipper could enable protons to pass along a wire of trapped water molecules across the hydrophobic membrane.

3D Hydrophobic Moment Vectors as a Tool to Characterize the Surface Polarity of Amphiphilic Peptides

Co-reporter:Sabine Reißer, Erik Strandberg, Thomas Steinbrecher, Anne S. Ulrich

Biophysical Journal (3 June 2014) Volume 106(Issue 11) pp:

Publication Date(Web):3 June 2014

DOI:10.1016/j.bpj.2014.04.020

The interaction of membranes with peptides and proteins is largely determined by their amphiphilic character. Hydrophobic moments of helical segments are commonly derived from their two-dimensional helical wheel projections, and the same is true for β-sheets. However, to the best of our knowledge, there exists no method to describe structures in three dimensions or molecules with irregular shape. Here, we define the hydrophobic moment of a molecule as a vector in three dimensions by evaluating the surface distribution of all hydrophilic and lipophilic regions over any given shape. The electrostatic potential on the molecular surface is calculated based on the atomic point charges. The resulting hydrophobic moment vector is specific for the instantaneous conformation, and it takes into account all structural characteristics of the molecule, e.g., partial unfolding, bending, and side-chain torsion angles. Extended all-atom molecular dynamics simulations are then used to calculate the equilibrium hydrophobic moments for two antimicrobial peptides, gramicidin S and PGLa, under different conditions. We show that their effective hydrophobic moment vectors reflect the distribution of polar and nonpolar patches on the molecular surface and the calculated electrostatic surface potential. A comparison of simulations in solution and in lipid membranes shows how the peptides undergo internal conformational rearrangement upon binding to the bilayer surface. A good correlation with solid-state NMR data indicates that the hydrophobic moment vector can be used to predict the membrane binding geometry of peptides. This method is available as a web application on http://www.ibg.kit.edu/HM/.

Structural Role of the Conserved Cysteines in the Dimerization of the Viral Transmembrane Oncoprotein E5

Co-reporter:Dirk Windisch, Silke Hoffmann, Sergii Afonin, Stefanie Vollmer, Soraya Benamira, Birgid Langer, Jochen Bürck, Claudia Muhle-Goll, Anne S. Ulrich

Biophysical Journal (22 September 2010) Volume 99(Issue 6) pp:

Publication Date(Web):22 September 2010

DOI:10.1016/j.bpj.2010.06.073

The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor β, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane environment and elucidate the role of the Cys-Ser-Cys motif, we produced recombinantly the wild-type protein and four cysteine mutants. Comparison by circular dichroism in detergent micelles and lipid vesicular dispersion and by NMR in trifluoroethanol demonstrates that the absence of one or both cysteines does not influence the highly α-helical secondary structure, nor does it impair the ability of E5 to dimerize, observations that are further supported by sodium dodecylsulfate polyacrylamide gel electrophoresis. We also observed assemblies of higher order. Oriented circular dichroism in lipid bilayers shows that E5 is aligned as a transmembrane helix with a slight tilt angle, and that this membrane alignment is also independent of any cysteines. We conclude that the Cys-containing motif represents a disordered region of the protein that serves as an extra covalent connection for stabilization.

Bilayer-Mediated Clustering and Functional Interaction of MscL Channels

Co-reporter:Stephan L. Grage, Asbed M. Keleshian, Tamta Turdzeladze, Andrew R. Battle, Wee C. Tay, Roland P. May, Stephen A. Holt, Sonia Antoranz Contera, Michael Haertlein, Martine Moulin, Prithwish Pal, Paul R. Rohde, V. Trevor Forsyth, Anthony Watts, Kerwyn Casey Huang, Anne S. Ulrich, Boris Martinac

Biophysical Journal (2 March 2011) Volume 100(Issue 5) pp:

Publication Date(Web):2 March 2011

DOI:10.1016/j.bpj.2011.01.023

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

Synergistic Insertion of Antimicrobial Magainin-Family Peptides in Membranes Depends on the Lipid Spontaneous Curvature

Co-reporter:Erik Strandberg, Jonathan Zerweck, Parvesh Wadhwani, Anne S. Ulrich

Biophysical Journal (19 March 2013) Volume 104(Issue 6) pp:

Publication Date(Web):19 March 2013

DOI:10.1016/j.bpj.2013.01.047

PGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state 15N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions.

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

Co-reporter:Dirk Windisch, Colin Ziegler, Stephan L. Grage, Jochen Bürck, Marcel Zeitler, Peter L. Gor’kov, Anne S. Ulrich

Biophysical Journal (18 August 2015) Volume 109(Issue 4) pp:

Publication Date(Web):18 August 2015

DOI:10.1016/j.bpj.2015.07.022

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 15N-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.

Conformation and Membrane Orientation of Amphiphilic Helical Peptides by Oriented Circular Dichroism

Co-reporter:Jochen Bürck, Siegmar Roth, Parvesh Wadhwani, Sergii Afonin, Nathalie Kanithasen, E. Strandberg, Anne S. Ulrich

Biophysical Journal (15 October 2008) Volume 95(Issue 8) pp:

Publication Date(Web):15 October 2008

DOI:10.1529/biophysj.108.136085

Oriented circular dichroism (OCD) was used to characterize and compare in a quantitative manner the secondary structure and concentration dependent realignment of the antimicrobial peptides PGLa and MSI-103, and of the structurally related cell-penetrating peptide MAP in aligned phospholipid bilayers. All these peptides adopt an amphiphilic α-helical conformation, and from solid-state NMR analysis they are known to bind to membranes in two distinct orientations depending on their concentration. At low peptide/lipid (P/L) ratio the helices are aligned parallel to membrane surface (S-state), but with increasing concentration they realign to a tilted orientation (T-state), getting immersed into the membrane with an oblique angle supposedly as a result of dimer-formation. In macroscopically aligned liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers the two limiting states are represented by distinct OCD spectra, and all spectra at intermediate peptide concentrations can be described by a linear combination of these two line shapes. The corresponding fraction of molecules occupying the T-state was determined by fitting the intermediate spectra with a superposition of the two extreme line shapes. By plotting this fraction versus 1/(P/L), the threshold P/L* ratio for realignment was extracted for each of the three related peptides. Despite their structural similarity distinctly different thresholds were obtained, namely for MSI-103 realignment starts already at a low P/L of ∼1:236, for a MAP derivative (using a nonaggregating analog containing a D-amino acid) the transition begins at P/L ∼1:156, whereas PGLa needs the highest concentration to flip into T-state at P/L ∼1:85. Analysis of the original MAP sequence (containing only L-amino acids) gave OCD spectra compatible with β-pleated conformation, suggesting that this peptide starts to aggregate with increasing concentration, unlike the other helical peptides. All these changes in peptide conformation and membrane alignment observed here by OCD seem to be functionally relevant, as they can be correlated with the membrane perturbing activities of the three antimicrobial and cell-penetrating sequences.

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

Co-reporter:Parvesh Wadhwani, Nico Heidenreich, Benjamin Podeyn, Jochen Bürck and Anne S. Ulrich

Biomaterials Science (2013-Present) 2017 - vol. 5(Issue 4) pp:NaN827-827

Publication Date(Web):2017/03/09

DOI:10.1039/C7BM00069C

γ-(S)-Trifluoromethyl proline: evaluation as a structural substitute of proline for solid state 19F-NMR peptide studies

Co-reporter:Vladimir Kubyshkin, Sergii Afonin, Sezgin Kara, Nediljko Budisa, Pavel K. Mykhailiuk and Anne S. Ulrich

Organic & Biomolecular Chemistry 2015 - vol. 13(Issue 11) pp:NaN3181-3181

Publication Date(Web):2015/02/13

DOI:10.1039/C5OB00034C

Calculation of fluorine chemical shift tensors for the interpretation of oriented 19F-NMR spectra of gramicidin A in membranes

Co-reporter:Ulrich Sternberg, Marco Klipfel, Stephan L. Grage, Raiker Witter and Anne S. Ulrich

Physical Chemistry Chemical Physics 2009 - vol. 11(Issue 32) pp:NaN7060-7060

Publication Date(Web):2009/07/20

DOI:10.1039/B908236K

Stereochemical effects on the aggregation and biological properties of the fibril-forming peptide [KIGAKI]3 in membranes

Physical Chemistry Chemical Physics 2013 - vol. 15(Issue 23) pp:NaN8971-8971

Publication Date(Web):2013/05/07

DOI:10.1039/C3CP50896J

Methyl (2S,4S)-1-Boc-4-trifluoromethylpyrrolidine-2-carboxylate

CAS:1523541-90-3

Methyl (2S,4S)-1-Boc-4-trifluoromethylpyrrolidine-2-carboxylate

L-Proline,L-valyl-L-arginyl-L-leucyl-L-prolyl-L-prolyl-L-prolyl-L-valyl-L-arginyl-L-leucyl-L-prolyl-L-prolyl-L-prolyl-L-valyl-L-arginyl-L-leucyl-L-prolyl-L-prolyl-

CAS:803732-09-4

L-Proline,L-valyl-L-arginyl-L-leucyl-L-prolyl-L-prolyl-L-prolyl-L-valyl-L-arginyl-L-leucyl-L-prolyl-L-prolyl-L-prolyl-L-valyl-L-arginyl-L-leucyl-L-prolyl-L-prolyl-

CAS:749875-10-3

(2S,4S)-1-(tert-Butoxycarbonyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid

CAS:470482-41-8

(2S,4S)-1-(tert-Butoxycarbonyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid

L-Isoleucinamide,L-lysyl-L-isoleucylglycyl-L-alanyl-L-lysyl-L-isoleucyl-L-lysyl-L-isoleucylglycyl-L-alanyl-L-lysyl-L-isoleucyl-L-lysyl-L-isoleucylglycyl-L-alanyl-L-lysyl-

CAS:355399-59-6

L-Isoleucinamide,L-lysyl-L-isoleucylglycyl-L-alanyl-L-lysyl-L-isoleucyl-L-lysyl-L-isoleucylglycyl-L-alanyl-L-lysyl-L-isoleucyl-L-lysyl-L-isoleucylglycyl-L-alanyl-L-lysyl-