Hybrid Amyloid-Based Redox Hydrogel for Bioelectrocatalytic H Oxidation.

An artificial amyloid-based redox hydrogel was designed for mediating electron transfer between a [NiFeSe] hydrogenase and an electrode. Starting from a mutated prion-forming domain of fungal protein HET-s, a hybrid redox protein containing a single benzyl methyl viologen moiety was synthesized. This protein was able to self-assemble into structurally homogenous nanofibrils.

Development of Conformational Antibodies to Detect Bcl-xL's Amyloid Aggregates in Metal-Induced Apoptotic Neuroblastoma Cells.

Bcl-xL, a member of the Bcl-2 family, is a pro-survival protein involved in apoptosis regulation. We have previously reported the ability of Bcl-xL to form various types of fibers, from native to amyloid conformations. Here, we have mimicked the effect of apoptosis-induced caspase activity on Bcl-xL by limited proteolysis using trypsin.

Magnetic Nanoparticles Applications for Amyloidosis Study and Detection: A Review.

Magnetic nanoparticles (MNPs) have great potential in biomedical and clinical applications because of their many unique properties. This contribution provides an overview of the MNPs mainly used in the field of amyloid diseases. The first part discusses their use in understanding the amyloid mechanisms of fibrillation, with emphasis on their ability to control aggregation of amyloidogenic proteins.

Mass and charge distributions of amyloid fibers involved in neurodegenerative diseases: mapping heterogeneity and polymorphism.

Heterogeneity and polymorphism are generic features of amyloid fibers with some important effects on the related disease development. We report here the characterization, by charge detection mass spectrometry, of amyloid fibers made of three polypeptides involved in neurodegenerative diseases: Aβ peptide, tau and α-synuclein. Beside the mass of individual fibers, this technique enables to characterize the heterogeneity and the polymorphism of the population.

Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting.

Aim: Gadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses.

Materials & Methods: The ability of the functionalized nanoparticles to target amyloid fibrils made of β-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy.

Results: Nanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with K values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles.

Interprotein Electron Transfer between FeS-Protein Nanowires and Oxygen-Tolerant NiFe Hydrogenase.

Self-assembled redox protein nanowires have been exploited as efficient electron shuttles for an oxygen-tolerant hydrogenase. An intra/inter-protein electron transfer chain has been achieved between the iron-sulfur centers of rubredoxin and the FeS cluster of [NiFe] hydrogenases. [NiFe] Hydrogenases entrapped in the intricated matrix of metalloprotein nanowires achieve a stable, mediated bioelectrocatalytic oxidation of H at low-overpotential.

A synthetic redox biofilm made from metalloprotein-prion domain chimera nanowires.

Engineering bioelectronic components and set-ups that mimic natural systems is extremely challenging. Here we report the design of a protein-only redox film inspired by the architecture of bacterial electroactive biofilms. The nanowire scaffold is formed using a chimeric protein that results from the attachment of a prion domain to a rubredoxin (Rd) that acts as an electron carrier.

Gd-nanoparticles functionalization with specific peptides for ß-amyloid plaques targeting.

Background: Amyloidoses are characterized by the extracellular deposition of insoluble fibrillar proteinaceous aggregates highly organized into cross-β structure and referred to as amyloid fibrils. Nowadays, the diagnosis of these diseases remains tedious and involves multiple examinations while an early and accurate protein typing is crucial for the patients' treatment. Routinely used neuroimaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) using Pittsburgh compound B, [(11)C]PIB, provide structural information and allow to assess the amyloid burden, respectively, but cannot discriminate between different amyloid deposits.

Mass Determination of Entire Amyloid Fibrils by Using Mass Spectrometry.

Amyloid fibrils are self-assembled protein structures with important roles in biology (either pathogenic or physiological), and are attracting increasing interest in nanotechnology. However, because of their high aspect ratio and the presence of some polymorphism, that is, the possibility to adopt various structures, their characterization is challenging and basic information such as their mass is unknown. Here we show that charge-detection mass spectrometry, recently developed for large self-assembled systems such as viruses, provides such information in a straightforward manner.

Biochemical and biophysical characterization of the selenium-binding and reducing site in Arabidopsis thaliana homologue to mammals selenium-binding protein 1.

The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants.

Real-time NMR characterization of structure and dynamics in a transiently populated protein folding intermediate.

Recent advances in NMR spectroscopy and the availability of high magnetic field strengths now offer the possibility to record real-time 3D NMR spectra of short-lived protein states, e.g., states that become transiently populated during protein folding.

Amyloid fibrils formed by the programmed cell death regulator Bcl-xL.

The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis.

Accessibility changes within diphtheria toxin T domain upon membrane penetration probed by hydrogen exchange and mass spectrometry.

The translocation domain of diphtheria toxin inserts in membrane and becomes functional when the pH inside endosomes is acid. At that stage, the domain is in a partially folded state; this prevents the use of high-resolution methods for the characterization of its functional structure. On that purpose, we report here the use of hydrogen/deuterium exchange experiments coupled to mass spectrometry.

Investigation at residue level of the early steps during the assembly of two proteins into supramolecular objects.

Understanding the driving forces governing protein assembly requires the characterization of interactions at molecular level. We focus on two homologous oppositely charged proteins, lysozyme and α-lactalbumin, which can assemble into microspheres. The assembly early steps were characterized through the identification of interacting surfaces monitored at residue level by NMR chemical shift perturbations by titrating one (15)N-labeled protein with its unlabeled partner.

Kinetics and structure during self-assembly of oppositely charged proteins in aqueous solution.

Self-assembly in aqueous solution of two oppositely charged globular proteins, hen egg white lysozyme (LYS) and bovine calcium-depleted α-lactalbumin (apo α-LA), was investigated at pH 7.5. The aggregation rate of equimolar mixtures of the two proteins was determined using static and dynamic light scattering as a function of the ionic strength (15-70 mM) and protein concentration (0.

Solution and membrane-bound chaperone activity of the diphtheria toxin translocation domain towards the catalytic domain.

During cell intoxication by diphtheria toxin, endosome acidification triggers the translocation of the catalytic (C) domain into the cytoplasm. This event is mediated by the translocation (T) domain of the toxin. Previous work suggested that the T domain acts as a chaperone for the C domain during membrane penetration of the toxin.

Accessibility changes within diphtheria toxin T domain when in the functional molten globule state, as determined using hydrogen/deuterium exchange measurements.

The translocation domain (T domain) of diphtheria toxin adopts a partially folded state, the so-called molten globule state, to become functional at acidic pH. We compared, using hydrogen/deuterium exchange experiments associated with MS, the structures of the T domain in its soluble folded state at neutral pH and in its functional molten globule state at acidic pH. In the native state, the alpha-helices TH5 and TH8 are identified as the core of the domain.

Characterization of the structure and intermolecular interactions between the connexin40 and connexin43 carboxyl-terminal and cytoplasmic loop domains.

Gap junctions are intercellular channels that allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. They are formed by two hemichannels, each constituted by the oligomerization of six connexins (Cx). Among the 21 different human Cx isoforms, studies have suggested that in the heart, Cx40 and Cx43 can oligomerize to form heteromeric hemichannels.

Deciphering membrane insertion of the diphtheria toxin T domain by specular neutron reflectometry and solid-state NMR spectroscopy.

Insertion and translocation of soluble proteins into and across biological membranes are involved in many physiological and pathological processes, but remain poorly understood. Here, we describe the pH-dependent membrane insertion of the diphtheria toxin T domain in lipid bilayers by specular neutron reflectometry and solid-state NMR spectroscopy. We gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane.

Membrane Interaction of botulinum neurotoxin A translocation (T) domain. The belt region is a regulatory loop for membrane interaction.

The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH.

Kinetics of fibril formation of bovine kappa-casein indicate a conformational rearrangement as a critical step in the process.

S-carboxymethylated (SCM) kappa-casein forms in vitro fibrils that display several characteristics of amyloid fibrils, although the protein is unrelated to amyloid diseases. In order to get insight into the processes that prevent the formation of amyloid fibrils made of kappa-caseins in milk, we have characterized in detail the reaction and the roles of its possible effectors: glycosylation and other caseins. Given that native kappa-casein occurs as a heterogeneous mixture of carbohydrate-free and carbohydrate-containing chains, kinetics of fibril formation were performed on purified glycosylated and unglycosylated SCM kappa-caseins using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and Fourier transform infrared spectroscopy for morphological and structural analyses.

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy.

Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event.

Concerted protonation of key histidines triggers membrane interaction of the diphtheria toxin T domain.

The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction.

Type III secretion system translocator has a molten globule conformation both in its free and chaperone-bound forms.

Type III secretion systems of Gram-negative pathogenic bacteria allow the injection of effector proteins into the cytosol of host eukaryotic cells. Crossing of the eukaryotic plasma membrane is facilitated by a translocon, an oligomeric structure made up of two bacterial proteins inserted into the host membrane during infection. In Pseudomonas aeruginosa, a major human opportunistic pathogen, these proteins are PopB and PopD.

Defining the interacting regions between apomyoglobin and lipid membrane by hydrogen/deuterium exchange coupled to mass spectrometry.

Sperm whale myoglobin can be considered as the model protein of the globin family. The pH-dependence of the interactions of apomyoglobin with lipid bilayers shares some similarities with the behavior of pore-forming domains of bacterial toxins belonging also to the globin family. Two different states of apomyoglobin bound to a lipid bilayer have been characterized by using hydrogen/deuterium exchange experiments and mass spectrometry.