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The TAR DNA-binding protein (TDP-43) self-assembles into prion-like aggregates considered to be the structural hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we use a combination of electron microscopy, X-ray fiber diffraction, Fourier-transform infrared spectroscopy analysis, and solid-state NMR spectroscopy to investigate the molecular organization of different TDP constructs, namely the full-length TDP-43 (1-414), two C-terminal fragments [TDP-35 (90-414) and TDP-16 (267-414)], and a C-terminal truncated fragment (TDP-43 increment GaroS2), in their fibrillar state. Although the different protein constructs exhibit similar fibril morphology and a typical cross-beta signature by X-ray diffraction, solid-state NMR indicates that TDP-43 and TDP-35 share the same polymorphic molecular structure, while TDP-16 encompasses a well-ordered amyloid core. We identified several residues in the so-called C-terminal GaroS2 (368-414) domain that participates in the rigid core of TDP-16 fibrils, underlining its importance during the aggregation process. Our findings demonstrate that C-terminal fragments can adopt a different molecular conformation in isolation or in the context of the full-length assembly, suggesting that the N-terminal domain and RRM domains play an important role in the TDP-43 amyloid transition.

International audience; The transient receptor potential ankyrin 1 (TRPA1) channel is a polymodal sensor of environmental irritant compounds, endogenous proalgesic agents, and cold. Upon activation, TRPA1 channels increase cellular calcium levels via direct permeation and trigger signaling pathways that hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP 2) in the inner membrane leaflet. Our objective was to determine the extent to which a putative PIP 2-interaction site (Y1006-Q1031) is involved in TRPA1 regulation. The interactions of two specific peptides (L992-N1008 and T1003-P1034) with model lipid membranes were characterized by biophysical approaches to obtain information about affinity, peptide secondary structure, and peptide effect in the lipid organization. The results indicate that the two peptides interact with lipid membranes only if PIP 2 is present and their affinities depend on the presence of calcium. Using whole-cell electrophysiology, we demonstrate that mutation at F1020 produced channels with faster activation kinetics and with a rightward shifted voltage-dependent activation curve by altering the allosteric constant that couples voltage sensing to pore opening. We assert that the presence of PIP 2 is essential for the interaction of the two peptide sequences with the lipid membrane. The putative phosphoinositide-interacting domain comprising the highly conserved F1020 contributes to the stabilization of the TRPA1 channel gate.

The morphology and secondary structure of peptide fibers formed by aggregation of tubulin-associated unit (Tau) fragments (K18), in the presence of the inner cytoplasmic membrane phosphatidylinositol component (PIP2) or heparin sodium (HS) as cofactors, are determined with nanoscale (

For the first time, natural Aβ1–42 fibrils (WT) implicated in Alzheimer's disease, as well as two synthetic mutants forming less toxic amyloid fibrils (L34T) and highly toxic oligomers (oG37C), are chemically characterized at the scale of a single structure using tip-enhanced Raman spectroscopy (TERS). While the proportion of TERS features associated with amino acid residues is similar for the three peptides, a careful examination of amide I and amide III bands allows us to clearly distinguish WT and L34T fibers organized in parallel β-sheets from the small and more toxic oG37C oligomers organized in anti-parallel β-sheets.

Nickel–iron hydrogenase ([NiFe] Hase) catalyzes hydrogen splitting into protons and electrons, and is a potential biocatalyst in fuel cells. Three FeS clusters aligned as a conductive wire drive electrons from the [NiFe] active site to the surface of the enzyme, where the redox partner (including the electrode) binds. Direct enzyme connection gave access to thermodynamic and kinetic data of enzymatic reactions through direct electron transfer (DET). Mediated electron transfer (MET) allowed recreation of the physiological electron-transfer chain, and/or connection of unfavorably oriented enzymes. Previous work demonstrated that DET or MET processes for H2 oxidation by a soluble, O2-sensitive [NiFe] Hase from Desulfovibrio species could be controlled by electrostatic interaction. The presence of an acidic patch of amino acids, coupled to a dipole moment pointing towards the distal FeS cluster (positioned at the surface of the enzyme), allowed orientation of the enzyme, which turned upside down as a function of the charge on the electrochemical interface. Recently, we reported on the electrochemistry of membrane-bound Aquifex aeolicus (Aa) [NiFe] Hase, which exhibits outstanding resistance to O2, CO, and heat. [8–10] Efficient immobilization of this Hase was achieved on graphite electrodes, in aqueous electrolytes and ionic liquids, by encapsulation in carbon nanotube networks, or connection to a redox polymer. In contrast to the soluble, O2sensitive [NiFe] Hase, no specific orientation could be obtained by electrostatic interaction for Aa Hase, and thus control of the electron-transfer process was not possible. A model structure accordingly put forward a very different environment of the distal FeS cluster, with no charged amino acid patch, in accordance with the membrane anchorage. We analyze herein H2 oxidation by Aa Hase immobilized on self-assembled monolayers (SAMs) on gold electrodes as a function of both the length and the nature of the thiol derivative (see SI 1 and SI 2 in the Supporting Information). For the first time, AFM and polarization modulation infrared reflection adsorption (PM-IRRA) studies are reported for understanding Aa Hase orientation and its consequences for electron-transfer process in H2 oxidation. Positively charged 4-aminothiophenol (ArNH2) and negatively charged 6-mercaptohexanoic acid (C5COOH) SAMs both yield DET and MET processes for H2 oxidation (Figure 1a and b), and neither process is favored over the other. A mixed process was similarly observed for H2 oxidation at charged short-chain alkanethiols, which are known to bemore disordered. This strongly suggests that electroenzymatic H2 oxidation is linked to multiple orientations of Hase on top of the charged SAMs, and not to Hase present inside possible SAM defects. The lipophilic methylene blue (MB) molecule

Direct identification of organic colourants in heterogeneous matrices of cultural heritage objects with very little or no sampling remains a challenging analytical task. A Raman procedure was investigated for the direct identification of archaeological organic-coloured pigments found in works of art, without solvent extraction. Conventional micro-Raman spectroscopy (MRS) using different excitations and surface-enhanced Raman scattering (SERS) were tested on microscopic samples of paints and cosmetics containing dyes from different sources (animal or vegetal). A tiny lump of purple pigment discovered during excavations in the ancient Minoan city of Akrotiri, on the Santorini Island in Greece (1650 B.C.) and Greco–Roman pink cosmetics were studied. In some cases the results were compared with a range of lake pigments made in the laboratory following historical recipes. Copyright © 2008 John Wiley & Sons, Ltd.

Raman spectroscopy was employed for the quantitative analysis of mixtures of the three dyes used at the Manufacture Nationale des Gobelins (Paris, France), fixed on wool fibres. It is based on a laboratory-written program that implements the decomposition of the combined Raman spectrum into the relative contributions of the spectra of each constituent. To perfect the method, it was first applied to fibres dyed at the laboratory and then extended to the dyeing baths, before and after the dyeing process. Binary and ternary mixtures, in which the quantities were known, were used. The percentages obtained by applying the quantitative analysis of the Raman spectra were similar to those introduced. The method therefore allows quantification of the dyes and gives information about the dyeing process. Fibres recently dyed at the workshop of the Manufacture Nationale des Gobelins with unknown proportions were analysed using this method. The results obtained are in conformity with the observed colour of the dyed hanks of wool. Copyright © 2006 John Wiley & Sons, Ltd.

The ability of DNA to bind polycation yielding polyplexes is widely used in nonviral gene delivery. The aim of the present study was to evaluate the DNA compaction with a new DNA vector using Raman spectroscopy. The polyplexes result from an association of a beta-cyclodextrin polymer (polybeta-CD), an amphiphilic cationic connector (DC-Chol or adamantane derivative Ada2), and DNA. The charge of the polymeric vector is effectively controlled by simple addition of cationic connector in the medium. We used surface enhanced Raman spectroscopy (SERS) to characterize this ternary complex, monitoring the accessibility of adenyl residues to silver colloids. The first experiments were performed using model systems based on polyA (polyadenosine monophosphate) well characterized by SERS. This model was then extended to plasmid DNA to study polybeta-CD/Ada2/DNA and polybeta-CD/DC-Chol/DNA polyplexes. The SERS spectra show a decrease of signal intensity when the vector/DNA charge ratio (Z+/-) increases. At the highest ratio (Z+/- = 10) the signal is 6-fold and 3-fold less intense than the DNA reference signal for Ada2 and DC-Chol polyplexes, respectively. Thus adenyl residues have a reduced accessibility as DNA is bound to the vector. Moreover, the SERS intensity variations are in agreement with gel electrophoresis and zeta potential experiments on the same systems. The overall study clearly demonstrates that the cationic charges neutralizing the negative charges of DNA result in the formation of stable polyplexes. In vitro transfection efficiency of those DNA vectors are also presented and compared to the classical DC-Chol lipoplexes (DC-Chol/DNA). The results show an increase of the transfection efficiency 2-fold higher with our vector based on polybeta-CD.

Surface-enhanced resonance Raman spectroscopy (SERRS) was employed to study the potential-dependent processes of the electron transferring heme protein cytochrome c552 (Cyt-c522) from Thermus thermophilus. Cyt-c552 was adsorbed on Ag electrodes coated with functionalized self-assembled monolayers (SAMs) of alkanethiols, regarded as a model of its redox partner ba3-oxidase. By a quantitative analysis of the SERR spectra recorded at different potentials, the redox potential of Cyt-c552 and the number of transferred electrons were determined. On pure hydrophobic alkanethiols, the Cyt-c552 heme structure is greatly modified in a non-electroactive way with the appearance of a 5cHS species. When Cyt-c552 is adsorbed on mixed surfaces of hydroxyl- and methyl-terminated alkanethiols, the electron transfer is effective (n = 1) and the structure of the heme is not modified, as is assumed when Cyt-c552 interacts with its natural redox partner ba3-oxidase. When the chain length of the mixed SAMs is increased, the defects on the surface are decreased and the electron transfer becomes less efficient. The presence of defects in the organization of the short chain of five carbons seems to be relevant for having a good surface model of the redox partner. Copyright © 2003 John Wiley & Sons, Ltd.

Invasive fungal infections are a major cause of mortality among solid organ transplant recipients. Scopulariopsis species and their teleomorph Microascus are molds found in soil and decaying organic matter. We report here the case of a woman who underwent bilateral lung transplantation for severe emphysema. On day 25 after transplantation, endobronchial green-black lesions were detected during routine endoscopy. Endobronchial swabs, biopsies, and bronchoalveolar lavage samples were positive for Microascus cirrosus. This fungal infection developed despite voriconazole given for previous persistent invasive aspergillosis. Treatment consisted of a combination of antifungal medication (voriconazole, terbinafine, amphotericin B, and caspofungin) and endoscopic resection of necrosed bronchial mucosa. A favorable clinical outcome was achieved after 7 weeks of treatment. Seven cases of Scopulariopsis/Microascus infection have been previously described in solid organ transplant recipients. Only two survived after treatment with an antifungal combination therapy including echinocandins, posaconazole, and terbinafine. In immunocompromised patients, infection by Microascus species is a rare but life-threatening event because of innate resistance to most common antifungal drugs. Our patient was successfully cured by combined therapy including intravenous voriconazole and caspofungin, oral terbinafine, and inhaled voriconazole and amphotericin B administered for 7 weeks in association with iterative endoscopic debridement to reduce fungal inoculum.

The structural changes of cytochrome c(552) bound to anionic and hydrophobic clay surfaces have been investigated by Fourier transform infrared spectroscopy. Binding to the anionic surface of montmorillonite is controlled by electrostatic interactions since addition of electrolyte (0.5 mol L(-1) KCl) causes desorption of more than 2/3 of the protein molecules. Electrostatic binding occurs through the back side of the protein (i.e., remote from the heme site) and is associated only with subtle changes of the secondary structure. In contrast, adsorption to the hydrophobic surface of talc leads to a decrease in alpha-helical structure by ca. 5% and an increase in beta-sheet structure by ca. 6%. These structural changes are attributed to a hydrophobic region on the front surface of cytochrome c(552) close to the partially exposed heme edge. This part on the protein surface is identified as the interaction domain for talc and most likely also serves for binding to the natural reaction partner, a ba(3)-oxidase. Fourier transform infrared spectra of cytochrome c(552) and the clay-cytochrome c(552) complexes have been measured as a function of time following dissolution and suspension in deuterated buffer, respectively. A two-dimensional correlation analysis was applied to these spectra to investigate the dynamics of the structural changes in the protein. For both complexes, adsorption and subsequent unfolding processes in the binding domains are faster than the time resolution of the spectroscopic experiments. Thus, the processes that could be monitored are refolding of peptide segments and side chain rearrangements following the adsorption-induced perturbation of the protein structure and the solvation of the adsorbed protein. In each case, side chain alterations of solvent-exposed tyrosine, aspartate, and glutamate residues were observed. For the cytochrome c(552)-talc complex, these changes are followed by a slow refolding of the peptide chain in the binding domain and, subsequently, a further H/D exchange of amide group protons.

One, two, and three coordinated phenoxyl radicals are formed by a series of differently substituted tris(phenolato)iron(III) complexes, [FeIIIL], which undergo three reversible one-electron oxidations. The cations [FeIIIL]+. and [FeIIIL]2+.. have been characterized by zero- and applied field Mossbauer and resonance Raman spectroscopy. Both techniques prove unambiguously and independently that these oxidations are ligand- rather than metal-centered processes.

Surface-enhanced Raman spectra (SERS at Creighton colloidal silver) and UV/visible spectra have been recorded for an antimicrobial agent (pefloxacin) at a biologically active concentration (ca. 10−6 mol/L−1). The adsorption of pefloxacin on the silver surface occurs both via the carboxylate group and the carbonyl of the pyridinone ring. The conjugated part of the molecule is tilted and gives rise to a charge transfer between the drug and the plasmon surface. However the orientation of the drug on the colloid varies with the concentration of pefloxacin, salt addition, and pH. Adsorption via only the carboxylate is privileged in presence of strongly competitive anions such as C1−. Thus the carbonyl of the pyridinone ring is desorbed, and the charge transfer is not detected. In basic medium the competitive OH− ion leads to similar orientation changes. For acidic pHs few residual molecules having a carboxylate function, or few carboxylic species bonded via the pyridinone CO group, remain adsorbed on the aggregated and unstable silver surface. The spectroscopic analyses and the measurements of the particle size of the colloid show that added salt increases the aggregation and enhances the pefloxacin SERS signals if the anion is not competitive. In the presence of NaNO3 an increase of the plasmon oscillations of the metal and a larger number of adsorption sites could explain the SERS amplitude. The competitive anions C1− and OH− to a lesser extent limit the colloid aggregation, pefloxacin adsorption, and SERS enhancement. The influence of the charge transfer on the Raman intensity appears to be weak. © 1995 John Wiley & Sons, Inc.