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B Bechinger

Publications and source records attributed to B Bechinger.

At least 19 recordsLinked to original sources
PubMed2001

Solid-state NMR investigations of interaction contributions that determine the alignment of helical polypeptides in biological membranes.

Helical peptides reconstituted into oriented phospholipid bilayers were studied by proton-decoupled 15N solid-state NMR spectroscopy. Whereas hydrophobic channel peptides, such as the N-terminal region of Vpu of HIV-1, adopt transmembrane orientations, amphipathic peptide antibiotics are oriented parallel to the bilayer surface. The interaction contributions that determine the alignment of helical peptides in lipid membranes were analysed using model sequences, and peptides that change their topology in a pH-dependent manner have been designed. The energy contributions of histidines, lysines, leucines and alanines as well as the alignment of peptides and phospholipids under conditions of hydrophobic mismatch have been investigated in considerable detail.

Amino Acid Sequence↗
PubMed2001

15N and 31P solid-state NMR investigations on the orientation of zervamicin II and alamethicin in phosphatidylcholine membranes.

The topologies of zervamicin II and alamethicin, labeled with (15)N uniformly, selectively, or specifically, have been investigated by oriented proton-decoupled (15)N solid-state NMR spectroscopy. Whereas at lipid-to-peptide (L/P) ratios of 50 (wt/wt) zervamicin II exhibits transmembrane alignments in 1,2-dicapryl (di-C10:0-PC) and 1,2-dilauroyl (di-C12:0-PC) phosphatidylcholine bilayers, it adopts orientations predominantly parallel to the membrane surface when the lengths of the fatty acyl chains are extended. The orientational order of zervamicin II increases with higher phospholipid concentrations, and considerable line narrowing is obtained in di-C10:0-PC/zervamicin II membranes at L/P ratios of 100 (wt/wt). In contrast to zervamicin, alamethicin is transmembrane throughout most, if not all, of its length when reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. The (31)P solid-state NMR spectra of all phospholipid/peptaibol samples investigated show a high degree of headgroup order, indicating that the peptides do not distort the bilayer structure. The observed differences in peptide orientation between zervamicin and alamethicin are discussed with reference to differences in their lengths, helical conformations, distribution of (hydroxy)proline residues, and hydrophobic moments. Possible implications for peptaibol voltage-gating are also described.

Alamethicin↗
PubMed2001

Calorimetric investigations of the structural stability and interactions of colicin B domains in aqueous solution and in the presence of phospholipid bilayers.

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the full-length toxin is consistent with two endothermic transitions. Whereas peak A (T(m) = 55 degrees C) most likely corresponds to the receptor/translocation domain, peak B (T(m) = 59 degrees C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 degrees C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T(m) = 65 degrees C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B ( approximately 40%) or its pore-forming fragment ( approximately 80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T(m) = 54 degrees C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.

Calorimetry↗
PubMed2000

Alignment of lysine-anchored membrane peptides under conditions of hydrophobic mismatch: a CD, 15N and 31P solid-state NMR spectroscopy investigation.

The secondary structure and alignment of hydrophobic model peptides in phosphatidylcholine membranes were investigated as a function of hydrophobic mismatch by CD and oriented proton-decoupled (15)N solid-state NMR spectroscopies. In addition, the macroscopic phase and the orientational order of the phospholipid headgroups was analyzed by proton-decoupled (31)P NMR spectroscopy. Both, variations in the composition of the polypeptide (10-30 hydrophobic residues) as well as the fatty acid acyl chain of the phospholipid (10-22 carbons) were studied. At lipid-to-peptide ratios of 50, the peptides adopt helical conformations and bilayer macroscopic phases are predominant. The peptide and lipid maintain much of their orientational order even when the peptide is calculated to be 3 A too short or 14 A too long to fit into the pure lipid bilayer. A continuous decrease in the (15)N chemical shift obtained from transmembrane peptides in oriented membranes suggests an increasing helical tilt angle when the membrane thickness is reduced. This response is, however, insufficient to account for the full hydrophobic mismatch. When the helix is much too long to span the membrane, both the lipid and the peptide order are perturbed, an indication of changes in the macroscopic properties of the membrane. In contrast, sequences that are much too short show little effect on the phospholipid headgroup order, but the peptides exhibit a wide range of orientational distributions predominantly close to parallel to the membrane surface. A thermodynamic formalism is applied to describe the two-state equilibrium between in-plane and transmembrane peptide orientations.

Alanine↗
PubMed2000

Membrane interactions and alignment of structures within the HIV-1 Vpu cytoplasmic domain: effect of phosphorylation of serines 52 and 56.

The cytoplasmic domain of the HIV-1 accessory protein Vpu is involved in the binding and degradation of the viral receptor CD4. In order to analyze previous structural models in the context of membrane environments, regions of Vpu(CYTO) incorporating particular conformational features have been synthesized and labelled with (15)N at selected backbone amides. Well-oriented proton-decoupled (15)N solid-state NMR spectra with (15)N chemical shifts at the most upfield position indicate that the amphipathic helix within [(15)N-Leu 45]-Vpu(27-57) strongly interacts with mechanically aligned POPC bilayers and adopts an orientation parallel to the membrane surface. No major changes in the topology of this membrane-associated amphipathic helix were observed upon phosphorylation of serine residues 52 and 56, although this modification regulates biological function of Vpu. In contrast, [(15)N-Ala 62]-Vpu(51-81) exhibits a pronounced (15)N chemical shift anisotropy.

Human Immunodeficiency Virus Proteins↗
PubMed2000

Refinement of the geometry of the retinal binding pocket in dark-adapted bacteriorhodopsin by heteronuclear solid-state NMR distance measurements.

The bacterial proton pump bacteriorhodopsin (BR) is a 26.5 kDa seven-transmembrane helical protein. Several structural models have been published at > or =1.55 A resolution. The initial cis-trans isomerization of the retinal moiety involves structural changes within <1 A. To understand the chromophore-protein interactions that are important for light-driven proton transport, very accurate measurements of the protein geometry are required. To reveal more structural details at the site of the retinal, we have, therefore, selectively labeled the tryptophan side chains of BR with (15)N and metabolically incorporated retinal, (13)C-labeled at position 14 or 15. Using these samples, heteronuclear distances were measured with high accuracy using SFAM REDOR magic angle spinning solid-state NMR spectroscopy in dark-adapted bacteriorhodopsin. This NMR technique is applied for the first time to a high-molecular mass protein. Two retinal conformers are distinguished by their different isotropic 14-(13)C chemical shifts. Whereas the C14 position of 13-cis-15-syn-retinal is 4.2 A from [indole-(15)N]Trp86, this distance is 3.9 A in the all-trans-15-anti conformer. This latter distance allows us to check on the details of the active center of BR in the various published models derived from X-ray and electron diffraction data. The experimental approach and the results reported in this paper enforce the notion that distances between residues of a membrane protein binding pocket and a bound ligand can be determined at subangstrom resolution.

Bacteriorhodopsins↗
PubMed2000

Grazing incidence X-ray diffraction of highly aligned phospholipid membranes containing the antimicrobial peptide magainin 2.

We present the first study of grazing incidence X-ray diffraction on a model system of phospholipid membranes and antimicrobial peptides. For this purpose, highly oriented multilamellar samples have been prepared on solid substrates. By this technique, the short-range order of the lipid chains in the fluid L(alpha) phase can be investigated quantitatively, including not only the mean distance between acyl chains, but also the associated correlation length. The short-range order in lecithin is found to be severely affected by the amphiphilic peptide magainin 2.

Amino Acid Sequence↗
PubMed2000

The topology of lysine-containing amphipathic peptides in bilayers by circular dichroism, solid-state NMR, and molecular modeling.

In order to better understand the driving forces that determine the alignment of amphipathic helical polypeptides with respect to the surface of phospholipid bilayers, lysine-containing peptide sequences were designed, prepared by solid-phase chemical synthesis, and reconstituted into membranes. CD spectroscopy indicates that all peptides exhibit a high degree of helicity in the presence of SDS micelles or POPC small unilamellar vesicles. Proton-decoupled (31)P-NMR solid-state NMR spectroscopy demonstrates that in the presence of peptides liquid crystalline phosphatidylcholine membranes orient well along glass surfaces. The orientational distribution and dynamics of peptides labeled with (15)N at selected sites were investigated by proton-decoupled (15)N solid-state NMR spectroscopy. Polypeptides with a single lysine residue adopt a transmembrane orientation, thereby locating this polar amino acid within the core region of the bilayer. In contrast, peptides with > or = 3 lysines reside along the surface of the membrane. With 2 lysines in the center of an otherwise hydrophobic amino acid sequence the peptides assume a broad orientational distribution. The energy of lysine discharge, hydrophobic, polar, and all other interactions are estimated to quantitatively describe the polypeptide topologies observed. Furthermore, a molecular modeling algorithm based on the hydrophobicities of atoms in a continuous hydrophilic-hydrophobic-hydrophilic potential describes the experimentally observed peptide topologies well.

Algorithms↗
PubMed2000

Understanding peptide interactions with the lipid bilayer: a guide to membrane protein engineering.

In recent years, studies of the interactions of designed hydrophobic and amphipathic polypeptides with biological membranes have progressed considerably. In-plane and transmembrane helical domains have been engineered as well as sequences that exhibit dynamic distributions of different topologies. These sequences not only help our understanding of the thermodynamic interaction contributions that determine peptide orientation, but also exhibit interesting biological functions as autonomous units. In addition, helices are considered to be folding intermediates of multi-spanning membrane proteins. The specificity and thermodynamic stability of transmembrane helix-helix interactions or the assembly of beta sheets at the membrane surface have, therefore, become a focus of ongoing research.

Animals↗
PubMed1999

The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy.

Linear peptide antibiotics have been isolated from amphibians, insects and humans and used as templates to design cheaper and more potent analogues for medical applications. Peptides such as cecropins or magainins are < or = 40 amino acids in length. Many of them have been prepared by solid-phase peptide synthesis with isotopic labels incorporated at selected sites. Structural analysis by solid-state NMR spectroscopy and other biophysical techniques indicates that these peptide antibiotics strongly interact with lipid membranes. In bilayer environments they exhibit amphipathic alpha-helical conformations and alignments of the helix axis parallel to the membrane surface. This contrasts the transmembrane orientations observed for alamethicin or gramicidin A. Models that have been proposed to explain the antibiotic and pore-forming activities of membrane-associated peptides, as well as other experimental results, include transmembrane helical bundles, wormholes, carpets, detergent-like effects or the in-plane diffusion of peptide-induced bilayer instabilities.

Alamethicin↗
PubMed1999

The interactions of histidine-containing amphipathic helical peptide antibiotics with lipid bilayers. The effects of charges and pH.

The alpha-helix of the designed amphipathic peptide antibiotic LAH(4 )(KKALLALALHHLAHLALHLALALKKA-NH(2)) strongly interacts with phospholipid membranes. The peptide is oriented parallel to the membrane surface under acidic conditions, but transmembrane at physiological pH (Bechinger, B. (1996) J. Mol. Biol. 263, 768-775). LAH(4) exhibits antibiotic activities against Escherichia coli and Bacillus subtilis; the peptide does not, however, lyse human red blood cells at bacteriocidal concentrations. The antibiotic activities of LAH(4) are 2 orders of magnitude more pronounced at pH 5 when compared with pH 7.5. Although peptide association at low pH is reduced when compared with pH 7.5, the release of the fluorophore calcein from large unilamellar 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol vesicles is more pronounced at pH values where LAH(4) adopts an orientation along the membrane surface. The calcein release experiments thereby parallel the results obtained in antibiotic assays. Despite a much higher degree of association, calcein release activity of LAH(4) is significantly decreased for negatively charged membranes. Pronounced differences in the interactions of LAH(4) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine membranes also become apparent when the mechanisms of dye release are investigated. The results presented in this paper support models in which antibiotic activity is caused by detergent-like membrane destabilization, rather than pore formation by helical peptides in transmembrane alignments.

Amino Acid Sequence↗
PubMed1999

Solution structure and orientation of the transmembrane anchor domain of the HIV-1-encoded virus protein U by high-resolution and solid-state NMR spectroscopy.

The structure of the membrane anchor domain (VpuMA) of the HIV-1-specific accessory protein Vpu has been investigated in solution and in lipid bilayers by homonuclear two-dimensional and solid-state nuclear magnetic resonance spectroscopy, respectively. Simulated annealing calculations, using the nuclear Overhauser enhancement data for the soluble synthetic peptide Vpu1-39 (positions Met-1-Asp-39) in an aqueous 2,2,2-trifluoroethanol (TFE) solution, afford a compact well-defined U-shaped structure comprised of an initial turn (residues 1-6) followed by a linker (7-9) and a short helix on the N-terminal side (10-16) and a further longer helix on the C-terminal side (22-36). The side chains of the two aromatic residues (Trp-22 and Tyr-29) in the longer helix are directed toward the center of the molecule around which the hydrophobic core of the folded VpuMA is positioned. As the observed solution structure is inconsistent with the formation of ion-conductive membrane pores defined previously for VpuMA in planar lipid bilayers, the isolated VpuMA domain as peptide Vpu1-27 was investigated in oriented phospholipid bilayers by proton-decoupled 15N cross polarization solid-state NMR spectroscopy. The line widths and chemical shift data of three selectively 15N-labeled peptides are consistent with a transmembrane alignment of a helical polypeptide. Chemical shift tensor calculations imply that the data sets are compatible with a model in which the nascent helices of the folded solution structure reassemble to form a more regular linear alpha-helix that lies parallel to the bilayer normal with a tilt angle of </=30 degrees. The arrangement of the membrane-associated structures described previously for the cytoplasmic domain and for the anchor domain of Vpu identified in this work is discussed.

Amino Acid Sequence↗
PubMed1999

Peptide structural analysis by solid-state NMR spectroscopy.

Solid-state nmr spectroscopy provides a robust method for investigating polypeptides that have been prepared by chemical synthesis and that are immobilized by strong interactions with solid surfaces or large macroscopic complexes. Solid-state nmr spectroscopy has been widely used to investigate membrane polypeptides or peptide aggregates such as amyloid fibrils. Whereas magic angle spinning solid-state nmr spectroscopy allows one to measure distances and dihedral angles with high accuracy, static membrane samples that are aligned with respect to the magnetic field direction allow one to determine the secondary structure of bound polypeptides and their orientation with respect to the bilayer normal. Peptide dynamics and the effect of polypeptides on the macroscopic phase preference of phospholipid membranes have been investigated in nonoriented samples. Investigations of the structure and topology of membrane channels, peptide antibiotics, signal sequences as well as model systems that allow one to dissect the interaction contributions in phospholipid membranes will be presented in greater detail.

Anti-Bacterial Agents↗
PubMed1999

The alignment of a voltage-sensing peptide in dodecylphosphocholine micelles and in oriented lipid bilayers by nuclear magnetic resonance and molecular modeling.

The S4 segments of voltage-gated sodium channels are important parts of the voltage-sensing elements of these proteins. Furthermore, the addition of the isolated S4 polypeptide to planar lipid bilayers results in stepwise increases of ion conductivity. In order to gain insight into the mechanisms of pore formation by amphipathic peptides, the structure and orientation of the S4 segment of the first internal repeat of the rat brain II sodium channel was investigated in the presence of DPC micelles by multidimensional solution NMR spectroscopy and solid-state NMR spectroscopy on oriented phospholipid bilayers. Both the anisotropic chemical shift observed by proton-decoupled (15)N solid-state NMR spectroscopy and the attenuating effects of DOXYL-stearates on TOCSY crosspeak intensities of micelle-associated S4 indicate that the central alpha-helical portion of this peptide is oriented approximately parallel to the membrane surface. Simulated annealing and molecular dynamics calculations of the peptide in a biphasic tetrachloromethane-water environment indicate that the peptide alpha-helix extends over approximately 12 residues. A less regular structure further toward the C-terminus allows for the hydrophobic residues of this part of the peptide to be positioned in the tetrachloromethane environment. The implications for possible pore-forming mechanisms are discussed.

Amino Acid Sequence↗
PubMed1999

Membrane helix orientation from linear dichroism of infrared attenuated total reflection spectra.

Oriented multilamellar systems containing phospholipids and peptides have been formed on a germanium internal reflection element. Attenuated total reflection infrared spectra have been recorded and the linear dichroism of peptide amide I and amide II bands measured. Using peptides for which the orientation had been previously studied under similar experimental conditions by 15N solid-state nuclear magnetic resonance spectroscopy, important conclusions were drawn on the approach to be used to derive secondary structure orientation in a membrane from dichroic ratios. In particular, it is shown that the influence of the film thickness and refractive index on the orientation determination can be evaluated from the value of RATRiso, i.e., the dichroic ratio of a dipole oriented at the magic angle or with isotropic mobility. A series of peptides was used to test the validity of our suggestions on various helix orientations in the membrane. These include magainin 2 and hydrophobic (hPhi20) model peptides, the transmembrane segment of glycophorin (GLY), and LAH4, a designed peptide antibiotic that changes between a transmembrane and an in-plane orientation in a pH-dependent manner.

Amino Acid Sequence↗
PubMed1998

Orientational distribution of alpha-helices in the colicin B and E1 channel domains: a one and two dimensional 15N solid-state NMR investigation in uniaxially aligned phospholipid bilayers.

Thermolytic fragments of the channel-forming bacterial toxins colicin B and colicin E1 were uniformly labeled with the 15N isotope and reconstituted into uniaxially oriented membranes. These well-aligned samples were investigated by proton-decoupled 15N solid-state NMR spectroscopy at 40.5 and 71.0 MHz. The one dimensional spectra indicate a predominant orientation of the colicin alpha-helices parallel to the bilayer surface but also the presence of a considerable proportion of peptide bonds that align in a transmembrane direction. The orientational distribution of 15N-labeled amide bonds is nearly identical for colicin B and E1, each a representative of a different group of membrane-active colicins. This comparison indicates common structural features of the water-soluble as well as the bilayer-associated proteins. When the pH is lowered, the orientational distribution of amide vectors exhibits only a small shift from in-plane to transmembrane orientations, in agreement with increased affinity and activity of colicins at acidic conditions. The 15N spectral line shape was independent of the bilayer phospholipid composition (100-75 mol % phosphatidylcholine/0-25 mol % phosphatidylglycerol) or the protein-to-lipid ratio in the range 1.7 - 12 wt %. Two dimensional separated local field spectroscopy (PISEMA) resolves almost 200 15N resonances of the colicin B channel protein. Approximately 50 15N signals resonate in a region characteristic of transmembrane helical residues, in strong support of the previously suggested umbrella conformation of the closed colicin channel.

Amino Acid Sequence↗
PubMed1998

Structure and dynamics of the antibiotic peptide PGLa in membranes by solution and solid-state nuclear magnetic resonance spectroscopy.

PGLa, a 21-residue member of the magainin family of antibiotic peptides, is shown to be helical between residues 6 and 21 when associated with detergent micelles by multidimensional solution nuclear magnetic resonance (NMR) spectroscopy. Solid-state NMR experiments on specifically 15N-labeled peptides in oriented phospholipid bilayer samples show that the helix axis is parallel to the plane of the bilayers. 15N solid-state NMR powder pattern line shapes obtained on unoriented samples demonstrate that the amino-terminal residues are highly mobile and that the fluctuations of backbone sites decrease from Ala6 toward the carboxy terminus. The powder pattern observed for 15N-labeled Ala20 is essentially that expected for a rigid site. These findings are similar to those for the 23-residue magainin2 peptide in membrane environments.

Amino Acid Sequence↗