Search

The heptad repeat 1 and 2 (HR1, HR2) regions in the spike protein of SARS-CoV 2 play a key role in the fusogenic mechanism of the virus with the host cell. During the fusion process they are thought to rearrange into an interdomain multimer. Functional fragments of the heptad repeat 1 and 2 regions in the spike protein of SARS-CoV 2 were chemically synthesized, labeled with nitrofurazone (NBD) and their interactions investigated by fluorescence spectroscopy. Steady state emission, fluorescence quenching, anisotropy and lifetime measurements in combination with a fluorophore dilution scheme were used to dissect multimer formation of HR1 and HR2 in quantitative detail. In addition, the investigation of the multimers by homo-FRET (via anisotropy) and lifetime measurements reveals new insights into the mechanism of fluorophore-fluorophore interactions in biological samples. [Display omitted] • The heptad repeat 1 region (fragment) of the spike protein of SARS-CoV 2 forms trimers in solution. • The heptad 2 region (fragment) binds to the heptad repeat 1 trimer in a cooperative manner to form a hetero-hexamer • Self-quenching as well as homo-FRET detects the formation of multimers. • Chromophore-Chromophore interactions are described by homo-FRET or the Exciton concept depending on their distance. [ABSTRACT FROM AUTHOR]

Solid-state NMR is a quickly developing technique that allows one to obtain structural information at atomic resolution in extended lipid bilayers in a rather unique manner. Two approaches have been developed for membrane proteins and peptides namely magic angle sample spinning and the use of uniaxially oriented membrane samples. The state-of-the-art of both approaches will be introduced and the perspectives of solid-state NMR spectroscopy in the context of other structural biology techniques, pressing biomedical questions and membrane biophysics will be discussed. [Display omitted] • Ultra-fast MAS solid-state NMR provides structures at high resolution. • 1H detected solid-state NMR works with small samples. • Oriented solid-state NMR reveals protein structures in extended lipid bilayers. [ABSTRACT FROM AUTHOR]

Abstract: 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, δ-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. [Copyright &y& Elsevier]

Abstract: 31P 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 31P 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 31P 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 31P 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

The gp41 type I membrane protein is part of the trimeric Env complex forming the spikes at the HIV surface. By interacting with cellular receptors, the Env protein complex initiates the infectious cycle of HIV. After the first contact has been established Env disassembles by shedding gp120 while the remaining gp41 undergoes a number of conformational changes which drive fusion of the cellular and the viral membranes. Here we investigated the membrane interactions and oligomerization of the two gp41 heptad repeat domains NHR and CHR. While these are thought to form a six-helix bundle in the post-fusion state little is known about their structure and role during prior fusion events. When investigated in aqueous buffer by CD and fluorescence quenching techniques the formation of NHR/CHR hetero-oligomers is detected. An equilibrium of monomers and hetero-oligomers is also observed in membrane environments. Furthermore, the partitioning to POPC or POPC/POPG 3/1 vesicles of the two domains alone or in combination has been studied. The membrane interactions were further characterized by 15N solid-state NMR spectroscopy of uniaxially oriented samples which shows that the polypeptide helices are oriented parallel to the bilayer surface. The 31P solid-state NMR spectra of the same samples are indicative of considerable disordering of the membrane packing. The data support models where NHR and CHR insert in the viral and cellular membranes, respectively, where they exhibit an active role in the membrane fusion events. Unlabelled Image • The heptad repeats of gp41form hetero-oligomers in solution and in membranes. • The CHR and NHR helices align parallel to the membrane surface. • The two domains cause membrane disordering. • CHR and NHR play an active role in membrane fusion. [ABSTRACT FROM AUTHOR]

The histidine-rich peptides of the LAH4 family were designed using cationic antimicrobial peptides such as magainin and PGLa as templates. The LAH4 amphipathic helical sequences exhibit a multitude of interesting biological properties such as antimicrobial activity, cell penetration of a large variety of cargo and lentiviral transduction enhancement. The parent peptide associates with lipid bilayers where it changes from an orientation along the membrane interface into a transmembrane configuration in a pH-dependent manner. Here we show that LAH4 adopts a transmembrane configuration in fully saturated DMPC membranes already at pH 3.5, i.e. much below the pK a of the histidines whereas the transition pH in POPC correlates closely with histidine neutralization. In contrast in POPG membranes the in-planar configuration is stabilized by about one pH unit. The differences in pH can be converted into energetic contributions for the in-plane to transmembrane transition equilibrium, where the shift in the transition pH due to lipid saturation corresponds to energies which are otherwise obtained by the exchange of several cationic with hydrophobic residues. A similar dependence on lipid saturation has also been observed when the PGLa and magainin antimicrobial peptides interact within lipid bilayers suggesting that the quantitative evaluation presented in this paper also applies to other membrane polypeptides. [Display omitted] • Lipid saturation strongly drives the interface-to-transmembrane realignment. • The energetic contribution of lipid saturation corresponds to removing numerous charges from the amino acid sequence. • Anionic lipids stabilize the interfacial localization of cationic amphipathic peptides. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

We present a structural study of biomimetic lipid bilayers interacting with the antimicrobial peptide magainin 2 amide, using grazing incidence X-ray diffraction and reciprocal space mapping (RSM) techniques. The short-range order of lipid chains in lecithin is found to be strongly reduced by the peptides. From the scattering intensity of the chain correlation peak, we can quantify the lateral length scale R over which the bilayer structure is affected by peptide binding. The non-local perturbation of the bilayer is discussed in the framework of bilayer elasticity theory. [Copyright &y& Elsevier]

NK-2 is an antimicrobial peptide derived from helices 3 and 4 of the pore-forming protein of natural killer cells, NK-lysin. It has potent activities against Gram-negative and Gram-positive bacteria, fungi and protozoan parasites without being toxic to healthy human cells. In biophysical assays its membrane activities were found to require phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), lipids which dominate the composition of bacterial membranes. Here the structure and activities of NK-2 in binary mixtures of different PE/PG composition were investigated. CD spectroscopy reveals that a threshold concentration of 50 % PG is needed for efficient membrane association of NK-2 concomitant with a random coil – helix transition. Association with PE occurs but is qualitatively different when compared to PG membranes. Oriented solid-state NMR spectroscopy of NK-2 specifically labelled with 15N indicates that the NK-2 helices are oriented parallel to the PG bilayer surface. Upon reduction of the PG content to 20 mol% interactions are weaker and/or an in average more tilted orientation is observed. Fluorescence spectroscopy of differently labelled lipids is in agreement of an interfacial localisation of both helices where the C-terminal end is in a less hydrophobic environment. By inserting into the membrane interface and interacting differently with PE and PG the peptides probably induce high curvature strain which result in membrane openings and rupture. [Display omitted] • The potent antimicrobial peptide NK-2 interacts differently with PE- and PG-rich lipid bilayers. • Both helices of the peptide exhibit an in-plane interfacial alignment in supported PG membranes. • This topology and differentiation between PG and PE results in structural stress and membrane openings. [ABSTRACT FROM AUTHOR]

Abstract γ-Secretase is an integral membrane protein complex and is involved in the cleavage of the amyloid precursor protein APP to produce amyloid-β peptides. Amyloid-β peptides are considered causative agents for Alzheimer's disease and drugs targeted at γ-secretase are investigated as therapeutic treatments. We synthesized new carprofen derivatives, which showed γ-secretase modulating activity and determined their precise position, orientation, and dynamics in lipid membranes by combining neutron diffraction, solid-state NMR spectroscopy, and molecular dynamics simulations. Our data indicate that the carprofen derivatives are inserted into the membrane interface, where the exact position and orientation depends on the lipid phase. This knowledge will help to understand the docking of carprofen derivatives to γ-secretase and in the design of new potent drugs. The approach presented here promises to serve as a general guideline how drug/target interactions in membranes can be analyzed in a comprehensive manner. Graphical abstract Unlabelled Image Highlights • Design and synthesis of new γ-secretase modulators based on carprofen scaffold • Characterization of position, orientation, and dynamics of γ-secretase modulators in membranes • General strategy to analyze drug/target interactions in membranes [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d -amino acids i.e. D Leu14 and D Ser17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all- l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d -amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity. [ABSTRACT FROM AUTHOR]

Abstract: SP-BCTERM is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-BCTERM exhibited a flexible topology controlled by subtle variations in lipid composition. SP-BCTERM-induced lipid realignment and/or conformational changes at the level of the head group were observed using 31P solid-state NMR spectroscopy. Measurements of the depth of SP-BCTERM indicated the peptide center positions ~8Å more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression. [Copyright &y& Elsevier]

Abstract: Biological membranes are characterized by a high degree of dynamics. In order to understand the function of membrane proteins and even more of membrane-associated peptides, these motional aspects have to be taken into consideration. Solid-state NMR spectroscopy is a method of choice when characterizing topological equilibria, molecular motions, lateral and rotational diffusion as well as dynamic oligomerization equilibria within fluid phase lipid bilayers. Here we show and review examples where the 15N chemical shift anisotropy, dipolar interactions and the deuterium quadrupolar splittings have been used to analyze motions of peptides such as peptaibols, antimicrobial sequences, Vpu, phospholamban or other channel domains. In particular, simulations of 15N and 2H-solid-state NMR spectra are shown of helical domains in uniaxially oriented membranes when rotation around the membrane normal or the helix long axis occurs. [Copyright &y& Elsevier]

In recent decades, several epimers of peptides containing d -amino acids have been identified in antimicrobial sequences, a feature which has been associated with post-translational modification. Generally, d -isomers present similar or inferior antimicrobial activity, only surpassing their epimers in resistance to peptidases. The naturally occurring l -Phenylseptin (l -Phes) and d -Phenylseptin (d -Phes) peptides (FFFDTLKNLAGKVIGALT- nh 2) were reported with d -epimer showing higher activity against Staphylococcus aureus and Xanthomonas axonopodis in comparison with the l -epimer. In this study, we combine structural (CD, solution NMR), orientational (solid-state NMR) and biophysical (ITC, DSC and DLS) studies to understand the role of the d -phenylalanine in the increase of the antimicrobial activity. Although both peptides are structurally similar in the helical region ranging from D4 to the C-terminus, significant structural differences were observed near the peptides' N-termini (which encompasses the FFF motif). Specific aromatic interactions involving the phenylalanine side chains of d -Phes is responsible to maintaining the F1-F3 residues on the hydrophobic face of the peptide, increasing its amphipathicity when compared to the l -epimer. The higher capability of d -Phes to exert an efficient anchoring in the hydrophobic core of the phospholipid bilayer indicates a pivotal role of the N-terminus in enhancing the interaction between the d -peptide and the membrane interface in relation to its epimer. [Display omitted] • Structural and biophysical investigations of the L and D-phenylseptins reveal details of the peptide-membrane interactions. • The role of the D-phenylalanine in the increase of the antimicrobial activity. • Interactions of phenylalanine side chains of D-phenylseptin strengthen the peptide amphipathicity and its membrane affinity. [ABSTRACT FROM AUTHOR]

The LAH4 family of amphipathic peptides exhibits pronounced antimicrobial, cell penetrating and nucleic acid transfection activities. Furthermore, variants were designed with potent lentiviral transduction enhancement. When viewed along a helical wheel the four histidines are arranged to form an amphipathic structure. In order to optimize some of these biological activities the number of leucine and alanine residues exposed to the hydrophilic surface was systematically varied which resulted in the design of vectofusin a peptide with strong lentiviral transduction enhancement activities. Here the series of peptides with varying numbers of alanine or leucine residues, respectively, framed by the histidines was tested for their calcein release activity. Interestingly, the membrane pore formation and DNA transfection activities show a clear correlation with the hydrophilic angle. In contrast the membrane partitioning and the propensity to adopt helical conformations was hardly affected as long as the hydrophilic angle did not exceed a limiting value of 150°. • Histidine-rich peptides have potent antimicrobial, cell penetration and transduction activities. • Membrane pore formation correlates with the hydrophilic angle of the amphipathic helical peptides • Transfection activity follows hydrophilic angle and pore formation. [ABSTRACT FROM AUTHOR]

Viral protein R (Vpr) is a small accessory protein of 96 amino acids that is present in Human and simian immunodeficiency viruses. Among the very different properties that Vpr possesses we can find cell penetrating capabilities. Based on this and on its capacity to interact with nucleic acids we previously investigated the DNA transfection properties of Vpr and subfragments thereof. We found that fragments of the C-terminal helical domain of Vpr are able to deliver efficiently plasmid DNA into different cell lines. As the amphipathic helix may play a role in the interactions with membranes, we investigated whether insertion of a proline residue in the α-helix modifies the transfection properties of Vpr. Unexpectedly, we found that the resulting Vpr55-82 Pro70 peptide was even more efficient than wild type Vpr55-82 in the gene delivery assays. Using circular dichroism, light scattering and solid-state NMR techniques, we characterized the secondary structure and interactions of Vpr and several mutants with model membranes. A model is proposed where the proline shifts the dissociation equilibrium of the peptide-cargo complex and thereby its endosomal release. Unlabelled Image • Viral protein R fragment Vpr55-82 displays high DNA transfection capabilities. • Replacement of isoleucine 70 in Vpr55-82 by a proline increases the transfection. • This Proline 70 mutation has little effect on the membrane alignment of the peptide. • The proline alters the oligomerization and helix propensity of the Vpr peptide. [ABSTRACT FROM AUTHOR]