Magnetically-orientable Tween-based model membranes for NMR studies of proteins.

We present a new membrane mimetic system using a membrane softening detergent commonly known as Tween 80 (TW80), to form oriented systems for solid-state NMR applications. TW80 is a fatty acid ester (oleate) of sorbitan polyethoxylate and a mild non-ionic surfactant. Phosphatidylcholine (PC)/TW80 model membrane systems were characterized by solid-state NMR and FTIR spectroscopy. 31P and 2H NMR spectra showed that DMPC (14:0) and DPPC (16:0) self-assemble with TW80 to form oriented structures, and maintain alignment over a wide range of molar ratios and temperatures. The addition of lanthanide ions revealed that the membrane alignment can be flipped from parallel to perpendicular with respect to the magnetic field direction. Using 15N solid-state NMR and a labeled model transmembrane peptide, we showed that TW80-based membranes can be employed to determine the peptide orientation in the magnetic field, which is useful for structural determination. Altogether, our work showed that TW80 could be exploited for direct and efficient membrane protein extraction and to enhance membrane and membrane protein orientation without using a detergent removal step. This approach could be extended to a wide range of membranes including native ones.

Structure of a Parkinson's Disease-Involved α-Synuclein Peptide Is Modulated by Membrane Composition and Physical State.

α-Synuclein (AS), the protein responsible for Parkinson's disease, contains a 12-residue-long sequence, AS71-82, that is thought to play a crucial role in the α-synuclein aggregation process. Neuronal membranes are direct interacting partners of α-synuclein and play a role in fibrillogenesis by providing a charged catalytic surface, notably from anionic phospholipids. However, details are lacking regarding the impact of membrane composition and the driving forces leading to membrane anchorage and peptide structure conversion. To decipher the interplay of α-synuclein with neuronal membranes, the structure of AS71-82 was investigated in the presence of anionic model membranes. Infrared (IR) spectroscopy and solid-state nuclear magnetic resonance data show that AS71-82 adopts a perfectly in-register parallel ß-sheet structure with fibrillar morphology upon interactions with anionic model membranes. IR thermotropism experiments conducted with several membrane compositions revealed that the phospholipids' phase transition induces a rearrangement of the AS71-82 ß-sheet structure. In contrast, membranes are not significantly affected by the presence of AS71-82, which advocates for the amyloid fibrils to lie loosely on the membrane surface. The results bring new arguments for the lipid-sensing capabilities of AS71-82 and revealed its protofibrillar structure. The striking similarities between AS71-82 and α-synuclein make it a potential good aggregation inhibitor upon chemical modifications.

Crown ether modified peptides: Length and crown ring size impact on membrane interactions.

Antimicrobial peptides are widely studied as an alternative to traditional antibiotics. However, they are difficult to develop, as multiple factors influence their potency and selectivity toward bacterial cells. In this paper, we investigate three simplified model peptides that bear crown ethers, and the effects of simple structural modifications (peptide length and crown ether ring size) on their secondary structures and their permeabilizing activity on living cells and model membranes made with egg yolk phosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol. Circular dichroism studies show that the peptide length and the crown ether ring size do influence the conformation, but no trend could be determined from the results. Permeabilization studies with model membranes and with red blood cells demonstrated that from 13 residues to 16 residues, there is a gradual increase in activity as the peptides get longer. However, the shortest tested analogs, with 12 residues, also exhibited an increase in activity caused by the removal of one amino acid that was bearing a crown ether. Permeabilization assays showed that larger ring size analogs showed higher hemolytic activities. Altogether, the results reported help design new and more selective antimicrobial peptides.

Vibrational Circular Dichroism Reveals Supramolecular Chirality Inversion of α-Synuclein Peptide Assemblies upon Interactions with Anionic Membranes.

Parkinson's disease is an incurable neurodegenerative disorder caused by the aggregation of α-synuclein (AS). This amyloid protein contains a 12-residue-long segment, AS71-82, that triggers AS pathological aggregation. This peptide is then essential to better understand the polymorphism and the dynamics of formation of AS fibrillar structures. In this work, vibrational circular dichroism showed that AS71-82 is random coil in solution and forms parallel ß-sheet fibrillar aggregates in the presence of anionic vesicles. Vibrational circular dichroism, with transmission electronic microscopy, revealed that the fibrillar structures exhibit a nanoscale tape-like morphology with a preferential supramolecular helicity. Whereas the structure handedness of some other amyloid peptides has been shown to be driven by pH, that of AS71-82 is controlled by peptide concentration and peptide-to-lipid (P:L) molar ratio. At low concentrations and low P:L molar ratios, AS71-82 assemblies have a left-twisted handedness, whereas at high concentrations and high P:L ratios, a right-twisted handedness is adopted. Left-twisted assemblies interconvert into right-twisted ones with time, suggesting a maturation of the amyloid structures. As fibril species with two chiralities have also been reported previously in Parkinson's disease Lewy bodies and fibrils, the present results seem relevant to better understand AS amyloid assembly and fibrillization in vivo. From a diagnosis or therapeutic point of view, it becomes essential that future fibril probes, inhibitors, or breakers target pathological assemblies with specific chirality and morphology, in particular, because they may change with the stage of the disease.

Understanding amyloid fibril formation using protein fragments: structural investigations via vibrational spectroscopy and solid-state NMR.

It is well established that amyloid proteins play a primary role in neurodegenerative diseases. Alzheimer's, Parkinson's, type II diabetes, and Creutzfeldt-Jakob's diseases are part of a wider family encompassing more than 50 human pathologies related to aggregation of proteins. Although this field of research is thoroughly investigated, several aspects of fibrillization remain misunderstood, which in turn slows down, or even impedes, advances in treating and curing amyloidoses. To solve this problem, several research groups have chosen to focus on short fragments of amyloid proteins, sequences that have been found to be of great importance for the amyloid formation process. Studying short peptides allows bypassing the complexity of working with full-length proteins and may provide important information relative to critical segments of amyloid proteins. To this end, efficient biophysical tools are required. In this review, we focus on two essential types of spectroscopic techniques, i.e., vibrational spectroscopy and its derivatives (conventional Raman scattering, deep-UV resonance Raman (DUVRR), Raman optical activity (ROA), surface-enhanced Raman spectroscopy (SERS), tip-enhanced Raman spectroscopy (TERS), infrared (IR) absorption spectroscopy, vibrational circular dichroism (VCD)) and solid-state nuclear magnetic resonance (ssNMR). These techniques revealed powerful to provide a better atomic and molecular comprehension of the amyloidogenic process and fibril structure. This review aims at underlining the information that these techniques can provide and at highlighting their strengths and weaknesses when studying amyloid fragments. Meaningful examples from the literature are provided for each technique, and their complementarity is stressed for the kinetic and structural characterization of amyloid fibril formation.

Progress in the synthesis of fluorinated phosphatidylcholines for biological applications.

The synthesis and biophysical studies of fluorinated phospholipids have attracted a lot of interest over the past 40 years. Mono- and polyfluorinated phospholipids, containing 1-4 fluorine atoms, are designed mostly with the goal of developing new model membranes. The fluorine atoms are herein used as probes, mainly in 19F-NMR spectroscopy, to study biomolecule complexes. In this case, the spectroscopic features of the fluorine atom and the preservation of the parent lipid properties are of primary importance. On the other hand, highly fluorinated phospholipids, which contain a perfluorinated segment in alkyl chains, are mainly developed as drug-delivery devices and oxygen carriers. Here, the particular chemical characteristics of fluorine are used to form more stable and less toxic lipid vesicles/emulsions. This review will focus on describing the synthetic pathways for mono-, poly- and highly fluorinated phosphatidylcholines. We will also discuss, though not thoroughly, their properties and potential applications.

Novel approaches to probe the binding of recoverin to membranes.

Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. We have investigated the interaction of recoverin with zwitterionic phosphatidylcholine bilayers, the major lipid component of the rod outer segment disk membranes, using both 31P and 19F solid-state nuclear magnetic resonance (NMR) and infrared spectroscopy. In particular, several novel approaches have been used, such as the centerband-only detection of exchange (CODEX) technique to investigate lipid lateral diffusion and 19F NMR to probe the environment of the recoverin myristoyl group. The results reveal that the lipid bilayer organization is not disturbed by recoverin. Non-myristoylated recoverin induces a small increase in lipid hydration that appears to be correlated with an increased lipid lateral diffusion. The thermal stability of recoverin remains similar in the absence or presence of lipids and Ca2+. Fluorine atoms have been strategically introduced at positions 4 or 12 on the myristoyl moiety of recoverin to, respectively, probe its behavior in the interfacial and more hydrophobic regions of the membrane. 19F NMR results allow the observation of the calcium-myristoyl switch, the myristoyl group experiencing two different environments in the absence of Ca2+ and the immobilization of the recoverin myristoyl moiety in phosphatidylcholine membranes in the presence of Ca2+.

Structural and Mechanical Roles for the C-Terminal Nonrepetitive Domain Become Apparent in Recombinant Spider Aciniform Silk.

Spider aciniform (or wrapping) silk is the toughest of the seven types of spider silks/glue due to a combination of high elasticity and strength. Like most spider silk proteins (spidroins), aciniform spidroin (AcSp1) has a large core repetitive domain flanked by relatively short N- and C-terminal nonrepetitive domains (the NTD and CTD, respectively). The major ampullate silk protein (MaSp) CTD has been shown to control protein solubility and fiber formation, but the aciniform CTD function remains unknown. Here, we compare fiber mechanical properties, solution-state structuring, and fibrous state secondary structural composition, and orientation relative to native aciniform silk for two AcSp1 repeat units with or without fused AcSp1- and MaSp-derived CTDs alongside three AcSp1 repeat units without a CTD. The native AcSp1 CTD uniquely modulated fiber mechanical properties, relative to all other constructs, directly correlating to a native-like structural transformation and alignment.

Membrane fluidity is a driving force for recoverin myristoyl immobilization in zwitterionic lipids.

Recoverin is the only protein for which the phenomenon of calcium-myristoyl switch has been demonstrated without ambiguity. It is located in rod disk membranes where the highest content in polyunsaturated lipid acyl chains can be found. However, although essential to better understand the inactivation of the phototransduction process, the role of membrane fluidity on recoverin recruitment is unclear. We have therefore investigated the immobilization of the recoverin myristoyl moiety in the presence of phosphocholine bilayers using 2H solid-state NMR spectroscopy. Several lipids with different acyl chains were selected to investigate model membranes characterized by different fluidity. Immobilization of the recoverin myristoyl moiety was successfully observed but only in the presence of calcium and in specific lipid disordered states, showing that an optimal fluidity is required for recoverin immobilization.

Membrane solid-state NMR in Canada: A historical perspective.

This manuscript presents an overview of more than 40years of membrane solid-state nuclear magnetic resonance (NMR) research in Canada. This technique is a method of choice for the study of the structure and dynamics of lipid bilayers; bilayer interactions with a variety of molecules such as membrane peptides, membrane proteins and drugs; and to investigate membrane peptide and protein structure, dynamics, and topology. Canada has a long tradition in this field of research, starting with pioneering work on natural and model membranes in the 1970s in a context of emergence of biophysics in the country. The 1980s and 1990s saw an emphasis on studying lipid structures and dynamics, and peptide-lipid and protein-lipid interactions. The study of bicelles began in the 1990s, and in the 2000s there was a rise in the study of membrane protein structures. Novel perspectives include using dynamic nuclear polarization (DNP) for membrane studies and using NMR in live cells. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

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

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

Amphiphilicity Is a Key Determinant in the Membrane Interactions of Synthetic 14-mer Cationic Peptide Analogues.

Cationic antimicrobial peptides are a component of the innate immune system of several organisms and represent an interesting alternative to fight multiresistant bacteria. In this context, we have elaborated a synthetic peptide scaffold allowing the study of the impact of different molecular determinants on the membrane interactions. The aim of the present study was to elucidate the mechanism of action of two cationic peptides that derive from a neutral 14-mer template peptide and where the hydrophilic portion is composed of a crown ether. The R5R10 peptide is active in the presence of both negatively charged and zwitterionic membranes (nonselective) and adopts an α-helical conformation, whereas the R4R11 peptide is more active in the presence of negatively charged membranes (selective) and forms intermolecular ß-sheet structures. Both the membrane topology and the location of the peptides have been assessed using solid-state NMR and attenuated total reflectance Fourier transform infrared spectroscopy. In addition, fluorescence experiments have been performed on different membrane mixtures to evaluate the ability of the peptides to induce a positive curvature to the membrane. Overall, for both the R5R10 and R4R11 peptides, the results are consistent with a mechanism of action similar to the sinking-raft model in which the peptides are mainly lying flat on the membrane surface and impose a bending stress to the membrane, thus leading to the formation of pores. Furthermore, the difference of membrane selectivity between R5R10 and R4R11 peptides is due to their differing amphipathic properties which modulate the membrane activity on zwitterionic model membranes.

Membrane Assembly and Ion Transport Ability of a Fluorinated Nanopore.

A novel 21-residue peptide incorporating six fluorinated amino acids was prepared. It was designed to fold into an amphiphilic alpha helical structure of nanoscale length with one hydrophobic face and one fluorinated face. The formation of a fluorous interface serves as the main vector for the formation of a superstructure in a bilayer membrane. Fluorescence assays showed this ion channel's ability to facilitate the translocation of alkali metal ions through a phospholipid membrane, with selectivity for sodium ions. Computational studies showed that a tetramer structure is the most probable and stable supramolecular assembly for the active ion channel structure. The results illustrate the possibility of exploiting multiple Fδ-:M+ interactions for ion transport and using fluorous interfaces to create functional nanostructures.

Major Ampullate Spider Silk with Indistinguishable Spidroin Dope Conformations Leads to Different Fiber Molecular Structures.

To plentifully benefit from its properties (mechanical, optical, biological) and its potential to manufacture green materials, the structure of spider silk has to be known accurately. To this aim, the major ampullate (MA) silk of Araneus diadematus (AD) and Nephila clavipes (NC) has been compared quantitatively in the liquid and fiber states using Raman spectromicroscopy. The data show that the spidroin conformations of the two dopes are indistinguishable despite their specific amino acid composition. This result suggests that GlyGlyX and GlyProGlyXX amino acid motifs (X = Leu, Glu, Tyr, Ser, etc.) are conformationally equivalent due to the chain flexibility in the aqueous environment. Species-related sequence specificity is expressed more extensively in the fiber: the ß-sheet content is lower and width of the orientation distribution of the carbonyl groups is broader for AD (29% and 58°, respectively) as compared to NC (37% and 51°, respectively). ß-Sheet content values are close to the proportion of polyalanine segments, suggesting that ß-sheet formation is mainly dictated by the spidroin sequence. The extent of molecular alignment seems to be related to the presence of proline (Pro) that may decrease conformational flexibility and inhibit chain extension and alignment upon drawing. It appears that besides the presence of Pro, secondary structure and molecular orientation contribute to the different mechanical properties of MA threads.

Discriminating Lipid- from Protein-Calcium Binding To Understand the Interaction between Recoverin and Phosphatidylglycerol Model Membranes.

Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. Understanding how calcium modulates these interactions and how it interacts with anionic lipid membranes is necessary to gain insights into the function of recoverin. In this work, infrared spectroscopy allowed us to show that the availability of calcium to recoverin is modulated by the presence of complexes involving phosphatidylglycerol (PG), which in turn regulates its interactions with this negatively charged lipid. Calcium can indeed be sequestered into strongly bound complexes with PG and is thus sparingly available to recoverin. The thermal stability of recoverin then decreases, which results in weakened interactions with PG. By contrast, when calcium is fully available to recoverin, the protein is thermally stable, indicating that it binds two calcium ions, which results in favorable interactions with negatively charged lipids. Consequently, the protein induces an increase in the chain-melting phase transition temperature of PG, which is indicative of an enhanced lipid chain packing resulting from the peripheral location of the protein. The secondary structure of recoverin is not affected by its interactions with anionic membrane lipids. Similar results have been obtained with saturated and unsaturated anionic lipids. This work shows that the recruitment of recoverin at the surface of anionic lipid membranes is dependent on the availability of calcium.

A 3D-psoriatic skin model for dermatological testing: The impact of culture conditions.

BACKGROUND: Inadequate representation of the human tissue environment during a preclinical screen can result in inaccurate predictions of compound effects. Consequently, pharmaceutical investigators are searching for preclinical models that closely resemble original tissue for predicting clinical outcomes. METHODS: The current research aims to compare the impact of using serum-free medium instead of complete culture medium during the last step of psoriatic skin substitute reconstruction. Skin substitutes were produced according to the self-assembly approach. RESULTS: Serum-free conditions have no negative impact on the reconstruction of healthy or psoriatic skin substitutes presented in this study regarding their macroscopic or histological appearances. ATR-FTIR results showed no significant differences in the CH2 bands between psoriatic substitutes cultured with or without serum, thus suggesting that serum deprivation did not have a negative impact on the lipid organization of their stratum corneum. Serum deprivation could even lead to a better organization of healthy skin substitute lipids. Percutaneous analyses demonstrated that psoriatic substitutes cultured in serum-free conditions showed a higher permeability to hydrocortisone compared to controls, while no significant differences in benzoic acid and caffeine penetration profiles were observed. CONCLUSIONS: Results obtained with this 3D-psoriatic skin substitute demonstrate the potential and versatility of the model. It could offer good prediction of drug related toxicities at preclinical stages performed in order to avoid unexpected and costly findings in the clinic. GENERAL SIGNIFICANCE: Together, these findings offer a new approach for one of the most important challenges of the 21st century, namely, prediction of drug toxicity.

Spider wrapping silk fibre architecture arising from its modular soluble protein precursor.

Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable "beads-on-a-string" concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/ß-sheet-containing fibre can be directly related to spidroin architecture and stability.

Mimicking and Understanding the Agglutination Effect of the Antimicrobial Peptide Thanatin Using Model Phospholipid Vesicles.

Thanatin is a cationic 21-residue antimicrobial and antifongical peptide found in the spined soldier bug Podisus maculiventris. It is believed that it does not permeabilize membranes but rather induces the agglutination of bacteria and inhibits cellular respiration. To clarify its mode of action, lipid vesicle organization and aggregation propensity as well as peptide secondary structure have been studied using different membrane models. Dynamic light scattering and turbidimetry results show that specific mixtures of negatively charged and zwitterionic phospholipid vesicles are able to mimic the agglutination effect of thanatin observed on Gram-negative and Gram-positive bacterial cells, while monoconstituent ("conventional") models cannot reproduce this phenomenon. The model of eukaryotic cell reveals no particular interaction with thanatin, which is consistent with the literature. Infrared spectroscopy shows that under the conditions under which vesicle agglutination occurs, thanatin exhibits a particular spectral pattern in the amide I' region and in the region associated with Arg side chains. The data suggest that thanatin mainly retains its hairpin structure, Arg residues being involved in strong interactions with anionic groups of phospholipids. In the absence of vesicle agglutination, the peptide conformation and Arg side-chain environment are similar to those observed in solution. The data show that a negatively charged membrane is required for thanatin to be active, but this condition is insufficient. The activity of thanatin seems to be modulated by the charge surface density of membranes and thanatin concentration.

Using infrared and Raman microspectroscopies to compare ex vivo involved psoriatic skin with normal human skin.

Psoriasis is a chronic dermatosis that affects around 3% of the world's population. The etiology of this autoimmune pathology is not completely understood. The barrier function of psoriatic skin is known to be strongly altered, but the structural modifications at the origin of this dysfunction are not clear. To develop strategies to reduce symptoms of psoriasis or adequate substitutes for modeling, a deep understanding of the organization of psoriatic skin at a molecular level is required. Infrared and Raman microspectroscopies have been used to obtain direct molecular-level information on psoriatic and healthy human skin biopsies. From the intensities and positions of specific vibrational bands, the lipid and protein distribution and the lipid order have been mapped in the different layers of the skin. Results showed a similar distribution of lipids and collagen for normal and psoriatic human skin. However, psoriatic skin is characterized by heterogeneity in lipid/protein composition at the micrometer scale, a reduction in the definition of skin layer boundaries and a decrease in lipid chain order in the stratum corneum as compared to normal skin. A global decrease of the structural organization is exhibited in psoriatic skin that is compatible with an alteration of its barrier properties.