Interactions and Insertion of Escherichia coli Hfq into Outer Membrane Vesicles as Revealed by Infrared and Orientated Circular Dichroism Spectroscopies.

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host-bacteria interactions which may not be defined solely by protein-protein and protein-outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs.

Determination of Secondary Structure of Proteins by Nanoinfrared Spectroscopy.

Nanoscale infrared spectroscopy (AFMIR) is becoming an important tool for the analysis of biological sample, in particular protein assemblies, at the nanoscale level. While the amide I band is usually used to determine the secondary structure of proteins in Fourier transform infrared spectroscopy, no tool has been developed so far for AFMIR. The paper introduces a method for the study of secondary structure of protein based on a protein library of 38 well-characterized proteins. Ascending stepwise linear regression (ASLR) and partial least square (PLS) regression were used to correlate spectrum characteristic bands with the major secondary structures (α-helixes and ß-sheets). ASLR appears to provide better results than PLS. The secondary structure predictions are characterized by a root mean square standard error in a cross validation of 6.39% for α-helixes and 6.23% for ß-sheets.

Unraveling Membrane Perturbations Caused by the Bacterial Riboregulator Hfq.

Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNAs. Previously, we showed that the Hfq C-terminal region forms an amyloid-like structure and that these fibrils interact with membranes. The immediate consequence of this interaction is a disruption of the membrane, but the effect on Hfq structure was unknown. To investigate details of the mechanism of interaction, the present work uses different in vitro biophysical approaches. We show that the Hfq C-terminal region influences membrane integrity and, conversely, that the membrane specifically affects the amyloid assembly. The reported effect of this bacterial master regulator on membrane integrity is discussed in light of the possible consequence on small regulatory RNA-based regulation.

Characterization of Bacterial Amyloids by Nano-infrared Spectroscopy.

Atomic force microscopy has been used for decades to study the topography of proteins during aggregation but with a lack of information on the secondary structure. On the contrary, infrared spectroscopy was able to study structural changes during the aggregation, but this analysis is complicated due to the presence of different species in mixtures and the poor spatial (~µm) resolution of the FTIR microscopy. Recently, Professor Alexandre Dazzi combined those techniques in the so-called AFM-IR. This method allows acquiring IR spectra at the nanometric scale and becomes a new standard method for the characterization of amyloid fibrils and, more generally, for the aggregation of proteins.

Analysis of Bacterial Amyloid Interaction with Lipidic Membrane by Orientated Circular Dichroism and Infrared Spectroscopies.

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and orientated circular dichroism (OCD) are complementary spectroscopies widely used for the analysis of protein samples such as the amyloids commonly renowned as neurodegenerative agents. Determining the secondary structure content of proteins, such as aggregated ß-sheets inside the amyloids and in various environments, including membranes and lipids, has made these techniques very valuable and complemental to high-resolution techniques such as nuclear magnetic resonance (NMR), X-ray crystallography, and cryo-electron microscopy. FTIR and CD are extremely sensitive to structural changes of proteins due to environmental changes. Furthermore, FTIR provides information on lipid modifications upon protein binding, whereas synchrotron radiation CD (SRCD) and OCD are sensitive to the subtle structural changes occurring in ß-sheet-rich proteins and their orientation or alignment with lipid bilayers. FTIR and CD techniques allow the identification of parallel and antiparallel ß-sheet content and are therefore complementary. In this chapter, we present FTIR and CD/OCD applications to study the interactions of bacterial amyloids with membranes and lipids. Moreover, we show how to decipher the spectroscopic signals to obtain information on the molecular structure of amyloids and their interaction with lipids, addressing potential amyloid insertion into membranes and the lipid bilayer adjustments observed.

Probing amyloid fibril secondary structures by infrared nanospectroscopy: experimental and theoretical considerations.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillary structure with a higher ß-sheet content than their native structure. Attenuated total reflection Fourier transform infrared spectroscopy only provides bulk analysis of a sample therefore it is impossible to discriminate between different aggregated structures. To overcome this limitation, near-field techniques like AFM-IR have emerged in the last twenty years to allow infrared nanospectroscopy. This technique obtains IR spectra with a spatial resolution of ten nanometres, the size of isolated fibrils. Here, we present essential practical considerations to avoid misinterpretations and artefacts during these analyses. Effects of polarization of the incident IR laser, illumination configuration and coating of the AFM probes are discussed, including the advantages and drawbacks of their use. This approach will improve interpretation of AFM-IR spectra especially for the determination of secondary structures of species not accessible using classical ATR-FTIR.

Ready-to-Use Germanium Surfaces for the Development of FTIR-Based Biosensors for Proteins.

Germanium is particularly suitable for the design of FTIR-based biosensors for proteins. The grafting of stable and thin organic layers on germanium surfaces remains, however, challenging. To tackle this problem, we developed a calix[4]arene-tetradiazonium salt decorated with four oligo(ethylene glycol) chains and a terminal reactive carboxyl group. This versatile molecular platform was covalently grafted on germanium surfaces to yield robust ready-to-use surfaces for biosensing applications. The grafted calixarene monolayer prevents nonspecific adsorption of proteins while allowing bioconjugation with biomolecules such as bovine serum albumin (BSA) or biotin. It is shown that the native form of the investigated proteins was maintained upon immobilization. As a proof of concept, the resulting calix[4]arene-based germanium biosensors were used through a combination of ATR-FTIR spectroscopy and fluorescence microscopy for the selective detection of streptavidin from a complex medium. This study opens real possibilities for the development of sensitive and selective FTIR-based biosensors devoted to the detection of proteins.

Characterization by Nano-Infrared Spectroscopy of Individual Aggregated Species of Amyloid Proteins.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillar structure with a higher ß-sheet content than in their native structure. To characterize them, we used an innovative tool that coupled infrared spectroscopy with atomic force microscopy (AFM-IR). With this method, we show that we can detect different individual aggregated species from oligomers to fibrils and study their morphologies by AFM and their secondary structures based on their IR spectra. AFM-IR overcomes the weak spatial resolution of usual infrared spectroscopy and achieves a resolution of ten nanometers, the size of isolated fibrils. We characterized oligomers, amyloid fibrils of Aß42 and fibrils of α-synuclein. To our surprise, we figured out that the nature of some surfaces (ZnSe) used to study the samples induces destructuring of amyloid samples, leading to amorphous aggregates. We strongly suggest taking this into consideration in future experiments with amyloid fibrils. More importantly, we demonstrate the advantages of AFM-IR, with a high spatial resolution (≤ 10 nm) allowing spectrum recording on individual aggregated supramolecular entities selected thanks to the AFM images or on thin layers of proteins.

Lipidated apolipoprotein E4 structure and its receptor binding mechanism determined by a combined cross-linking coupled to mass spectrometry and molecular dynamics approach.

Apolipoprotein E (apoE) is a forefront actor in the transport of lipids and the maintenance of cholesterol homeostasis, and is also strongly implicated in Alzheimer's disease. Upon lipid-binding apoE adopts a conformational state that mediates the receptor-induced internalization of lipoproteins. Due to its inherent structural dynamics and the presence of lipids, the structure of the biologically active apoE remains so far poorly described. To address this issue, we developed an innovative hybrid method combining experimental data with molecular modeling and dynamics to generate comprehensive models of the lipidated apoE4 isoform. Chemical cross-linking combined with mass spectrometry provided distance restraints, characterizing the three-dimensional organization of apoE4 molecules at the surface of lipidic nanoparticles. The ensemble of spatial restraints was then rationalized in an original molecular modeling approach to generate monomeric models of apoE4 that advocated the existence of two alternative conformations. These two models point towards an activation mechanism of apoE4 relying on a regulation of the accessibility of its receptor binding region. Further, molecular dynamics simulations of the dimerized and lipidated apoE4 monomeric conformations revealed an elongation of the apoE N-terminal domain, whereby helix 4 is rearranged, together with Arg172, into a proper orientation essential for lipoprotein receptor association. Overall, our results show how apoE4 adapts its conformation for the recognition of the low density lipoprotein receptor and we propose a novel mechanism of activation for apoE4 that is based on accessibility and remodeling of the receptor binding region.

Grafting of Oligo(ethylene glycol)-Functionalized Calix[4]arene-Tetradiazonium Salts for Antifouling Germanium and Gold Surfaces.

Biosensors that can determine protein concentration and structure are highly desired for biomedical applications. For the development of such biosensors, the use of Fourier transform infrared (FTIR) spectroscopy with the attenuated internal total reflection (ATR) configuration is particularly attractive, but it requires appropriate surface functionalization of the ATR optical element. Indeed, the surface has to specifically interact with a target protein in close contact with the optical element and must display antifouling properties to prevent nonspecific adsorption of other proteins. Here, we report robust monolayers of calix[4]arenes bearing oligo(ethylene glycol) (oEG) chains, which were grafted on germanium and gold surfaces via their tetradiazonium salts. The formation of monolayers of oEGylated calix[4]arenes was confirmed by AFM, IR, and contact angle measurements. The antifouling properties of these modified surfaces were studied by ATR-FTIR spectroscopy and fluorescence microscopy, and the nonspecific absorption of bovine serum albumin was found to be reduced by 85% compared to that of unmodified germanium. In other words, the organic coating by oEGylated calix[4]arenes provides remarkable antifouling properties, opening the way for the design of germanium- or gold-based biosensors.

ATR-FTIR Analysis of Amyloid Proteins.

Attenuated total reflection FTIR (ATR-FTIR) has been used for decades to study protein secondary structures. More recently, it reveals also to be an exquisite and sensitive tool to study and discriminate amyloid aggregates. Based on the analysis of specific spectral features of ß-sheet structures, we present here a detailed protocol to differentiate oligomers vs. fibrils. This protocol, applicable to all amyloid proteins, demonstrates the power of this inexpensive, rapid, and low protein material-demanding method.

Controlled Functionalization of Gold Nanoparticles with Mixtures of Calix[4]arenes Revealed by Infrared Spectroscopy.

Labile ligands such as thiols and carboxylates are commonly used to functionalize AuNPs, though little control over the composition is possible when mixtures of ligands are used. It was shown recently that robustly functionalized AuNPs can be obtained through the reductive grafting of calix[4]arenes bearing diazonium groups on the large rim. Here, we report a calix[4]arene-tetradiazonium decorated by four oligo(ethylene glycol) chains on the small rim, which upon grafting gave AuNPs with excellent stability thanks to the C-Au bonds. Mixtures of this calixarene and one with four carboxylate groups were grafted on AuNPs. The resulting particles were analyzed by infrared spectroscopy, which revealed that the composition of the ligand shell clearly reflected the ratio of calixarenes that was present in solution. This strategy opens the way to robustly protected AuNPs with well-defined numbers of functional or postfunctionalizable groups.

Distinct Mechanisms Determine α-Synuclein Fibril Morphology during Growth and Maturation.

Amyloid polymorphs have become one of the focal points of molecular studies of neurodegenerative diseases like Parkinson's disease. Due to their distinct biochemical properties and prion-like characteristics, insights into the molecular origin and stability of amyloid polymorphs over time are crucial for understanding the potential role of amyloid polymorphism in these diseases. Here, we systematically study the fibrillization of recombinantly produced human α-synuclein (αSyn) over an extended period of time to unravel the origin and temporal evolution of polymorphism. We follow morphological changes in the same fibril sample with atomic force microscopy over a period of 1 year. We show that wild-type (wt) αSyn fibrils undergo a slow maturation over time after reaching the plateau phase of aggregation (as detected in a Thioflavin-T fluorescence assay). This maturation, visualized by changes in the fibril periodicity over time, is absent in the disease mutant fibrils. The ß-sheet content of the plateau phase and matured fibrils, obtained using Fourier transform infrared spectroscopy, is however similar for the αSyn protein sequences, suggesting that the morphological changes in wt αSyn fibrils are tertiary or quaternary in origin. Furthermore, results from a reversibility assay show that the plateau phase fibrils do not disassemble over time. Together, the observed changes in the periodicity distributions and stability of the fibrillar core over time point toward two distinct mechanisms that determine the morphology of wt αSyn fibrils: competitive growth between different polymorphs during the fibrillization phase followed by a process wherein fibrils undergo slow maturation or annealing.

Structural Characterization of the Amyloid Precursor Protein Transmembrane Domain and Its γ-Cleavage Site.

Alzheimer's disease is the most common form of dementia that affects about 50 million of sufferers worldwide. A major role for the initiation and progression of Alzheimer's disease has been associated with the amyloid ß-peptide (Aß), which is a protease cleavage product of the amyloid precursor protein. The amyloid precursor protein is an integral membrane protein with a single transmembrane domain. Here, we assessed the structural integrity of the transmembrane domain within oriented phosphatidylcholine lipid bilayers and determined the tilt angle distribution and dynamics of various subdomains using solid-state NMR and attenuated total reflectance Fourier transform infrared spectroscopies. Although the overall secondary structure of the transmembrane domain is α-helical, pronounced conformational and topological heterogeneities were observed for the γ- and, to a lesser extent, the ζ-cleavage site, with pronounced implications for the production of Aß and related peptides, the development of the disease, and pharmaceutical innovation.

Structural remodeling during amyloidogenesis of physiological Nα-acetylated α-synuclein.

The misfolding and aggregation of the presynaptic protein α-synuclein (AS) into amyloid fibrils is pathognomonic of Parkinson's disease, though the mechanism by which this structural conversion occurs is largely unknown. Soluble oligomeric species that accumulate as intermediates in the process of fibril formation are thought to be highly cytotoxic. Recent studies indicate that oligomer-to-fibril AS transition plays a key role in cell toxicity and progression of neurodegeneration. We previously demonstrated that a subgroup of oligomeric AS species are ordered assemblies possessing a well-defined pattern of intermolecular contacts which are arranged into a distinctive antiparallel ß-sheet structure, as opposed to the parallel fibrillar fold. Recently, it was demonstrated that the physiological form of AS is N-terminally acetylated (Ac-AS). Here, we first showed that well-characterized conformational ensembles of Ac-AS, namely monomers, oligomers and fibrils, recapitulate many biophysical features of the nonacetylated protein, such as hydrodynamic, tinctorial, structural and membrane-leakage properties. Then, we relied on ATR-FTIR spectroscopy to explore the structural reorganization during Ac-AS fibrillogenesis. We found that antiparallel ß-sheet transient intermediates are built-up at early stages of aggregation, which then evolve to parallel ß-sheet fibrils through helix-rich/disordered species. The results are discussed in terms of regions of the protein that might participate in this structural rearrangement. Our work provides new insights into the complex conformational reorganization occurring during Ac-AS amyloid formation.

Reversing the peptide sequence impacts on molecular surface behaviour.

The protein's primary structure has all the information for specific protein/peptide folding and, in many cases, can define specific amphiphilic regions along molecules that are important for interaction with membranes. In order to shed light on how peptide sequence is important for the surface properties of amphiphilic peptides, we designed three pairs of peptides with the following characteristics: (1) all molecules have the same hydrophobic residues; (2) the couples differ from each other in their hydrophilic amino acids: positively, negatively and non-charged; (3) each pair has the same residues (same global molecular hydrophobicity) but the primary structure is reversed in comparison to its partner (retro-isomer), giving a molecule with a hydrophilic N or C-terminus and a hydrophobic C or N-terminus. Using the Langmuir monolayer approach, we observed that sequence reversal has a central role in the lateral stability of peptide monolayers, in the ability of the molecules to partition into the air-water interface and in the rheological properties of peptide films, whereas the peptide's secondary structure, determined by ATR-FTIR, was the same for all peptides. Reversing the sequence also gives a differential way of peptide/lipid interaction when peptides are in the presence of POPC lipid bilayers. Our results show how sequence inversion confers a distinctive peptide surface behaviour and lipid interaction for molecules with a similar structure.

Amyloid fibrils are the molecular trigger of inflammation in Parkinson's disease.

Parkinson's disease (PD) is an age-related movement disorder characterized by a progressive degeneration of dopaminergic neurons in the midbrain. Although the presence of amyloid deposits of α-synuclein (α-syn) is the main pathological feature, PD brains also present a severe permanent inflammation, which largely contributes to neuropathology. Although α-syn has recently been implicated in this process, the molecular mechanisms underlying neuroinflammation remain unknown. In the present study, we investigated the ability of different α-syn aggregates to trigger inflammatory responses. We showed that α-syn induced inflammation through activation of Toll-like receptor 2 (TLR2) and the nucleotide oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome only when folded as amyloid fibrils. Oligomeric species, thought to be the primary species responsible for the disease, were surprisingly unable to trigger the same cascades. As neuroinflammation is a key player in PD pathology, these results put fibrils back to the fore and rekindles discussions about the primary toxic species contributing to the disease. Our data also suggest that the inflammatory properties of α-syn fibrils are linked to their intrinsic structure, most probably to their cross-ß structure. Since fibrils of other amyloids induce similar immunological responses, we propose that the canonical fibril-specific cross-ß structure represents a new generic motif recognized by the innate immune system.

Two distinct ß-sheet structures in Italian-mutant amyloid-beta fibrils: a potential link to different clinical phenotypes.

Most Alzheimer's disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aß) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aß mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development of early-onset familial AD. This type of AD is associated with a younger age at disease onset, increased ß-amyloid accumulation, and Aß deposition in cerebral blood vessel walls, giving rise to cerebral amyloid angiopathy (CAA). It remains largely unknown how the Italian mutation results in the clinical phenotype that is characteristic of CAA. We therefore investigated how this single point mutation may affect the aggregation of Aß1-42 in vitro and structurally characterized the resulting fibrils using a biophysical approach. This paper reports that wild-type and Italian-mutant Aß both form fibrils characterized by the cross-ß architecture, but with distinct ß-sheet organizations, resulting in differences in thioflavin T fluorescence and solvent accessibility. E22K Aß1-42 oligomers and fibrils both display an antiparallel ß-sheet structure, in comparison with the parallel ß-sheet structure of wild-type fibrils, characteristic of most amyloid fibrils described in the literature. Moreover, we demonstrate structural plasticity for Italian-mutant Aß fibrils in a pH-dependent manner, in terms of their underlying ß-sheet arrangement. These findings are of interest in the ongoing debate that (1) antiparallel ß-sheet structure might represent a signature for toxicity, which could explain the higher toxicity reported for the Italian mutant, and that (2) fibril polymorphism might underlie differences in disease pathology and clinical manifestation.

Chemical cross-linking/mass spectrometry maps the amyloid ß peptide binding region on both apolipoprotein E domains.

Apolipoprotein E (apoE) binds the amyloid ß peptide (Aß), one of the major culprits in Alzheimer's disease development. The formation of apoE:Aß complexes is implicated in both Aß clearance and fibrillization. However, the binding interface between apoE and Aß is poorly defined despite substantial previous research efforts, and the exact role of apoE in the pathology of Alzheimer's disease remains largely elusive. Here, we compared the three main isoforms of apoE (E2, E3, and E4) for their interaction with Aß1-42 in an early stage of aggregation and at near physiological conditions. Using electron microscopy and Western blots, we showed that all three isoforms are able to prevent Aß fibrillization and form a noncovalent complex, with one molecule of Aß bound per apoE. Using chemical cross-linking coupled to mass spectrometry, we further examined the interface of interaction between apoE2/3/4 and Aß. Multiple high-confidence intermolecular apoE2/3/4:Aß cross-links confirmed that Lys16 is located in the region of Aß binding to apoE2/3/4. Further, we demonstrated that both N- and C-terminal domains of apoE2/3/4 are interacting with Aß. The cross-linked sites were mapped onto and evaluated in light of a recent structure of apoE. Our results support binding of the hydrophobic Aß at the apoE domain-domain interaction interface, which would explain how apoE is able to stabilize Aß and thereby prevent its subsequent aggregation.

The structural organization of the N-terminus domain of SopB, a virulence factor of Salmonella, depends on the nature of its protein partners.

The TTSS is used by Salmonella and many bacterial pathogens to inject virulence factors directly into the cytoplasm of target eukaryotic cells. Once translocated these so-called effector proteins hijack a vast array of crucial cellular functions to the benefit of the bacteria. In the bacterial cytoplasm, some effectors are stabilized and maintained in a secretion competent state by interaction with specific type III chaperones. In this work we studied the conformation of the Chaperone Binding Domain of the effector named Salmonella Outer protein B (SopB) alone and in complex with its cognate chaperone SigE by a combination of biochemical, biophysical and structural approaches. Our results show that the N-terminus part of SopB is mainly composed by α-helices and unfolded regions whose organization/stabilization depends on their interaction with the different partners. This suggests that the partially unfolded state of this N-terminal region, which confers the adaptability of the effector to bind very different partners during the infection cycle, allows the bacteria to modulate numerous host cells functions limiting the number of translocated effectors.