Biomedical subjects

Burkhard Bechinger

Publications and source records attributed to Burkhard Bechinger.

At least 19 recordsLinked to original sources

PubMed2006

Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy.

Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide bonds and domains relative to the bilayer normal. Using this approach the (15)N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the (2)H solid-state NMR spectra acquired from peptides labelled with 3,3,3-(2)H(3)-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium NMR line shapes are highly sensitive to small variations in the alignment of the C(alpha)-C(beta) bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic helices. Thereafter investigations of the viral channel peptides Vpu(1-27) and Influenza A M2(22-46) are shown. Whereas the (15)N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously been performed.

Deuterium↗

PubMed2006

Characterization of the gene transfer process mediated by histidine-rich peptides.

Peptides have an interesting potential as gene transfer agents not only because they are biodegradable but also due to their reduced size and the facility of product characterization and large-scale production. Recently, we have shown that the cationic amphipathic 26-mer peptide LAH4 possesses high DNA delivery capacities. To better understand its way of functioning and to further improve its efficiency, we synthesized and tested various LAH4 derivatives. The evaluation of their DNA delivery capacity, as well as their transfection efficiency, makes accessible valuable information about structure-function correlations. In particular, our results indicate the threshold peptide concentration for endosomal escape and conformational preferences for the peptide. Furthermore, the results indicate that a fine balance of peptide-DNA interactions is responsible for the high transfection activity of LAH4. Taken together, the data also suggest that the peptides efficiently transport DNA into the cytoplasm and that the creation of more potent transfection compounds probably needs improvement of other steps during the process.

Cell Line↗

PubMed2006

Detergent-like actions of linear amphipathic cationic antimicrobial peptides.

Antimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, delta-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be 'special cases' within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies.

Antimicrobial Cationic Peptides↗

PubMed2006

Structure and dynamics of membrane-associated ICP47, a viral inhibitor of the MHC I antigen-processing machinery.

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.

Cell Membrane↗

PubMed2006

Cationic amphipathic histidine-rich peptides for gene delivery.

Besides being a useful tool in research, gene transfer has a high potential as treatment for a variety of genetic and acquired diseases. However, in order to enable a gene to become a pharmaceutical, efficient and safe methods of delivery have to be developed. We recently found that cationic amphipathic histidine-rich peptide antibiotics can efficiently deliver DNA into mammalian cells. Our lead compound, LAH4 (KKALLALALHHLAHLALHLALALKKA), demonstrated in vitro transfection efficiencies comparable to those of commercially available reagents. Synthesis and evaluation of LAH mutants provided evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Moreover, recent results suggest that binding of the DNA complexes to the plasma membrane is mediated by heparan sulfate proteoglycans and that anionic phospholipids may be involved in the endosomal destabilization process. Finally, we also describe in this review the rationale that led to the development of LAH4 as a DNA carrier as well as the biophysical methods that have allowed us to propose a model which could explain the way this peptide destabilizes the endosomal bilayer.

Amino Acid Sequence↗

PubMed2006

Interactions involved in the realignment of membrane-associated helices. An investigation using oriented solid-state NMR and attenuated total reflection Fourier transform infrared spectroscopies.

A series of histidine-containing peptides (LAH4X6) was designed to investigate the membrane interactions of selected side chains. To this purpose, their pH-dependent transitions from in-plane to transmembrane orientations were investigated by attenuated total reflection Fourier transform infrared and oriented solid-state NMR spectroscopies. Peptides of the same family have previously been shown to exhibit antibiotic and DNA transfection activities. Solution NMR spectroscopy indicates that these peptides form amphipathic helical structures in membrane environments, and the technique was also used to characterize the pK values of all histidines in the presence of detergent micelles. Whereas one face of the amphipathic helix is clearly hydrophobic, the opposite side is flanked by four histidines surrounding six leucine, alanine, glycine, tryptophan, or tyrosine residues, respectively. This diversity in peptide composition causes pronounced shifts in the midpoint pH of the in-plane to transmembrane helical transition, which is completely abolished for the peptides carrying the most hydrophilic amino acid residues. These properties open up a conceptually new approach to study in a quantitative manner the hydrophobic as well as specific interactions of amino acids in membranes. Notably, the resulting scale for whole residue transitions from the bilayer interface to the hydrophobic membrane interior is obtained from extended helical sequences in lipid bilayers.

Alanine↗

PubMed2006

Proton-decoupled 15N and 31P solid-state NMR investigations of the Pf3 coat protein in oriented phospholipid bilayers.

The coat proteins of filamentous phage are first synthesized as transmembrane proteins and then assembled onto the extruding viral particles. We investigated the transmembrane conformation of the Pseudomonas aeruginosa Pf3 phage coat protein using proton-decoupled 15N and 31P solid-state NMR spectroscopy. The protein was either biochemically purified and uniformly labelled with 15N or synthesized chemically and labelled at specific sites. The proteins were then reconstituted into oriented phospholipid bilayers and the resulting samples analysed. The data suggest a model in which the protein adopts a tilted helix with an angle of approximately 30 degrees and an N-terminal 'swinging arm' at the membrane surface.

Capsid Proteins↗

PubMed2006

Enhanced membrane disruption and antibiotic action against pathogenic bacteria by designed histidine-rich peptides at acidic pH.

The histidine-rich amphipathic cationic peptide LAH4 has antibiotic and DNA delivery capabilities. Here, we explore the interaction of peptides from this family with model membranes as monitored by solid-state (2)H nuclear magnetic resonance and their antibiotic activities against a range of bacteria. At neutral pH, the membrane disruption is weak, but at acidic pH, the peptides strongly disturb the anionic lipid component of bacterial membranes and cause bacterial lysis. The peptides are effective antibiotics at both pH 7.2 and pH 5.5, although the antibacterial activity is strongly affected by the change in pH. At neutral pH, the LAH peptides were active against both methicillin-resistant and -sensitive Staphylococcus aureus strains but ineffective against Pseudomonas aeruginosa. In contrast, the LAH peptides were highly active against P. aeruginosa in an acidic environment, as is found in the epithelial-lining fluid of cystic fibrosis patients. Our results show that modest antibiotic activity of histidine-rich peptides can be dramatically enhanced by inducing membrane disruption, in this case by lowering the pH, and that histidine-rich peptides have potential as future antibiotic agents.

Amino Acid Sequence↗

PubMed2005

The antibiotic and DNA-transfecting peptide LAH4 selectively associates with, and disorders, anionic lipids in mixed membranes.

The histidine-rich amphipathic peptide LAH4 has antibiotic and DNA delivery capabilities. The peptide has a strong affinity for anionic lipids found in the outer membrane of bacterial membranes. A role for anionic lipids in release of cationic plasmid-containing complexes has been proposed previously, and disruption of membrane asymmetry and presentation of phosphatidylserine (PS) in the membrane outer leaflet is a general feature observed in diseased mammalian cells. Therefore, to understand the peptide-lipid interactions in more detail, solid-state NMR experiments on model membranes have been performed. 31P MAS NMR on mixed phosphatidylcholine (PC)/PS and PC/phosphatidylglycerol (PG) membranes has been used to demonstrate a strong interaction between LAH4 and anionic lipids. By using deuterated lipids and wide-line 2H NMR when probing lipid chain order, it is demonstrated that LAH4 preferentially interacts with PS over PC and effectively disorders the anionic PS lipid fatty acyl chains. In addition, we demonstrate that the efficiency of gene transfer in vitro to different cell lines is closely related to the degree of disruption of PS acyl chains for four isomers of LAH4. This work suggests a mechanism of selective destabilization by LAH4 of anionic lipids in the membranes of cells during transfection with implications for nucleic acid delivery in vivo.

Amino Acid Sequence↗

PubMed2005

Topological equilibria of ion channel peptides in oriented lipid bilayers revealed by 15N solid-state NMR spectroscopy.

Ion channel peptides have been prepared by solid-phase peptide synthesis, labeled with 15N at selected sites, and reconstituted into oriented lipid bilayers. The (Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2 peptide has previously been shown to exhibit well-defined and discrete ionic conductances when investigated by single-channel measurements [Lear, J. D., et al. (1988) Science 240, 1177]. Proton-decoupled 15N solid-state NMR spectroscopy indicates that (Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2 preferentially aligns parallel to the membrane surface in excellent agreement with its amphipathic helical structure. However, by carefully choosing the conditions of the membrane environment, significant contributions that are indicative of transmembrane alignments become obvious in the 15N chemical shift solid-state NMR spectra. The data thereby provide experimental evidence for an equilibrium between in-plane and transmembrane-oriented helix configurations where the transmembrane and surface-oriented peptide fractions are in slow exchange. Similar topological equilibria are observed when the N-terminus of the LS21 peptide is acetylated. These observations provide experimental support for previous models, suggesting that the channels observed in single-channel conductance measurements are indeed formed by hexameric transmembrane helical bundles. In contrast, the shorter peptide (Leu-Ser-Ser-Leu-Leu-Ser-Leu)2-CONH2 is oriented parallel to the membrane surface under all conditions tested. This peptide exhibits erratic conductance changes when investigated by electrophysiological methods, probably because it is too short to span the lipid bilayer.

Ion Channels↗

PubMed2005

Detergent-like properties of magainin antibiotic peptides: a 31P solid-state NMR spectroscopy study.

(31)P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in (31)P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid (31)P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type (31)P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.

Anisotropy↗

PubMed2004

Investigations of polypeptide rotational diffusion in aligned membranes by 2H and 15N solid-state NMR spectroscopy.

Transmembrane and in-plane oriented peptides have been prepared by solid-phase peptide synthesis, labeled with 3,3,3-2H3-alanine and 15N-leucine at two selected sites, and reconstituted into oriented phophatidylcholine membranes. Thereafter, proton-decoupled 15N and 2H solid-state NMR spectroscopy at sample orientations of the membrane normal parallel to the magnetic field direction have been used to characterize the tilt and rotational pitch angle of these peptides in some detail. In a second step the samples have been tilted by 90 degrees . In this setup the spectral line shapes are sensitive indicators of the rate of rotational diffusion. Whereas monomeric transmembrane peptides exhibit spectral averaging and well-defined resonances, larger complexes are characterized by broad spectral line shapes. In particular the deuterium line shape is sensitive to association of a few transmembrane helices. In contrast, the formation of much larger complexes affects the 15N chemical shift spectrum. The spectra indicate that in liquid crystalline membranes an amphipathic peptide of 14 amino acids exhibits fast rotational diffusion on both the 2H and 15N time scales (>10(-5) s). Extending the sequences to 26 amino acids results in pronounced changes of the 2H solid-state NMR spectrum, whereas the signal intensities of 15N solid-state NMR spectra degrade. Below the phase transition temperature of the phospholipid bilayers, motional averaging on the time scale of the 2H solid-state NMR spectrum ceases for transmembrane and in-plane oriented peptides. Furthermore at temperatures close to the phase transition the total signal intensities of the deuterium solid-state NMR spectra strongly decrease.

Alanine↗

PubMed2004

The alignment, structure and dynamics of membrane-associated polypeptides by solid-state NMR spectroscopy.

Solid-state NMR spectroscopy is being developed at a fast pace for the structural investigation of immobilized and non-crystalline biomolecules. These include proteins and peptides associated with phospholipid bilayers. In contrast to solution NMR spectroscopy, where complete or almost complete averaging leads to isotropic values, the anisotropic character of nuclear interactions is apparent in solid-state NMR spectra. In static samples the orientation dependence of chemical shift, dipolar or quadrupolar interactions, therefore, provides angular constraints when the polypeptides have been reconstituted into oriented membranes. Furthermore, solid-state NMR spectroscopy of aligned samples offers distinct advantages in allowing access to dynamic processes such as topological equilibria or rotational diffusion in membrane environments. Alternatively, magic angle sample spinning (MAS) results in highly resolved NMR spectra, provided that the sample is sufficiently homogenous. MAS spinning solid-state NMR spectra allow to measure distances and dihedral angles with high accuracy. The technique has recently been developed to selectively establish through-space and through-bond correlations between nuclei, similar to the approaches well-established in solution-NMR spectroscopy.

Anisotropy↗

PubMed2004

Tilt and rotational pitch angle of membrane-inserted polypeptides from combined 15N and 2H solid-state NMR spectroscopy.

Knowledge of the alignment of alpha-helical polypeptides with respect to the membrane surface and their dynamics in the membrane are key to understanding the functional mechanisms of channels, antibiotics, and signal or translocation peptides. In this paper polypeptides have been labeled with [3,3,3-(2)H(3)]alanine as well as with (15)N at single site amide positions and reconstituted into oriented phospholipid bilayers. A transmembrane and two amphipathic helical polypeptides with the deuterium label at orthogonal positions have been investigated by deuterium and proton-decoupled (15)N solid-state NMR spectroscopy. The (15)N chemical shift measurements and the deuterium quadrupole splitting exhibit a highly complementary functional dependence with respect to the spatial alignment of the polypeptide. Therefore, the combination of these two measurements allows one to determine both the tilt and the rotational pitch angle with high precision. In addition, the deuterium line shape is very sensitive to mosaic spread and the relative orientation of the peptide. The solid-state NMR measurements indicate that the model sequences exhibit a small degree of mosaicity, when at the same time the phospholipid headgroup region is significantly distorted. Furthermore, the (2)H solid-state NMR spectra reveal small orientational and dynamic differences when the fatty acyl chain composition of the phosphatidylcholine bilayers is modified.

Alanine↗

PubMed2004

Peptide-related alterations of membrane-associated water: deuterium solid-state NMR investigations of phosphatidylcholine membranes at different hydration levels.

Deuterated water associated with oriented POPC bilayers was investigated before and after the addition of 2 mol% peptide. Membranes in the presences of antimicrobial-(LAH4), pore-forming- (the segments M2 of influenza A and S4 of the domain I of rat brain sodium channels) or lysine-containing model peptides (LAK1 and LAK3) were investigated by (2)H and proton-decoupled (31)P solid-state NMR. The NMR spectra were recorded as a function of hydration in the range between 15 and 93% relative humidity and of sample composition. In the presence of peptides an increased association of water is observed. A quantitative analysis suggests that the peptide-induced changes in the lipid bilayer packing have a significant effect on membrane-water association. The quadrupolar splittings of (2)H(2)O at a given degree of hydration indicate that the changes of the water deuterium order parameter are specific for the peptide sequence and the lipid composition.

Amino Acid Sequence↗

PubMed2003

Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells.

Gene delivery has shown potential in a wide variety of applications, including basic research, therapies for genetic and acquired diseases, and vaccination. Most available nonviral systems have serious drawbacks such as the inability to control and scale the production process in a reproducible manner. Here, we demonstrate a biotechnologically feasible approach for gene delivery, using synthetic cationic amphipathic peptides containing a variable number of histidine residues. Gene transfer to different cell lines in vitro was achieved with an efficiency comparable to commercially available reagents. We provide evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Endosomal acidification is also required. Interestingly, even when complexed to DNA these peptides maintain a high level of antibacterial activity, opening the possibility of treating the genetic defect and the bacterial infections associated with cystic fibrosis with a single compound. Thus, this family of peptides represents a new class of agents that may have broad utility for gene transfer and gene therapy applications.

Amino Acid Sequence↗

PubMed2003

Deuterium solid-state NMR investigations of exchange labeled oriented purple membranes at different hydration levels.

Oriented purple membranes were equilibrated under controlled (2)H(2)O relative humidity ranging from 15% to 93% and introduced into the magnetic field of an NMR spectrometer with the membrane normal parallel to the magnetic field direction. Deuterium solid-state NMR spectra of these samples resolved four deuteron populations. Deuterons that have exchanged with amide protons of the protein exhibited a broad spectral line shape (<150 kHz). Furthermore, a broadened signal of deuterons tightly associated with protein and lipid is detected at low hydration, as well as two additional water populations that were present when the samples were equilibrated at >/=75% relative humidity. These latter ones are characterized by narrow quadrupolar splittings (<2.5 kHz) and orientation-dependent chemical shifts. Their deuterium relaxation times, measured as a function of temperature, indicate correlation times in the fast regime (10(-10) s) and activation energies of 13 kJ/mol (at 86% relative humidity). Differences in T(1) and T(2) relaxation together with small residual quadrupole splittings show that the mobility of the deuterons is anisotropic. The occurrence of these mobile water populations at high levels of purple membrane hydration (>/=75% relative humidity) correlate with proton pumping activity of bacteriorhodopsin, the fast kinetics of M-decay in the bacteriorhodopsin photocycle, and structural alterations of the protein during the M-state, which have been described previously.

Bacteriorhodopsins↗

PubMed2003

Biosynthesis of isotopically labeled gramicidins and tyrocidins by Bacillus brevis.

The three-dimensional structure of bilayer-associated gramicidin A is available from a structural data base. This and related peptides are, therefore, ideal model compounds to use during the implementation and development of new NMR techniques for the structural investigations of membrane proteins. As these methods rely on the isotopic labelling of single, selected or all sites, we have, investigated and optimised biochemical protocols using different strains of the Gram-positive bacterium Bacillus brevis. With newly developed schemes for isotopic labelling large amounts of gramicidin and tyrocidin enriched with stable isotopes such as (15)N or (15)N/(13)C have been obtained at low cost. A variety of analytical and spectroscopic techniques, including HPLC, mass spectrometry and NMR spectroscopy are used to characterise the resulting products.

Amino Acid Sequence↗