Insights on the dynamic behavior of protein disulfide isomerase in the solution environment through the SAXS technique.

Unlabelled: The dynamic behavior of Protein Disulfide Isomerase (PDI) in an aqueous solution environment under physiologically active pH has been experimentally verified in this study using Small Angle X-ray Scattering (SAXS) technique. The structural mechanism of dimerization for full-length PDI molecules and co-complex with two renowned substrates has been comprehensively discussed. The structure models obtained from the SAXS data of PDI purified from bovine liver display behavior duality between unaccompanied-enzyme and after engaged with substrates.

Smad7 in intestinal CD4 T cells determines autoimmunity in a spontaneous model of multiple sclerosis.

Environmental triggers acting at the intestinal barrier are thought to contribute to the initiation of autoimmune disorders. The transforming growth factor beta inhibitor Smad7 determines the phenotype of CD4 T cells. We hypothesized that Smad7 in intestinal CD4 T cells controls initiation of opticospinal encephalomyelitis (OSE), a murine model of multiple sclerosis (MS), depending on the presence of gut microbiota.

Structural Insights into Curli CsgA Cross-β Fibril Architecture Inspire Repurposing of Anti-amyloid Compounds as Anti-biofilm Agents.

Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curli subunit, CsgA, revealing steric zipper fibrils of tightly mated β-sheets, demonstrating a structural link between curli and human pathological amyloids. D-enantiomeric peptides, originally developed to interfere with Alzheimer's disease-associated amyloid-β, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium.

Expression, purification and stabilization of human serotonin transporter from E. coli.

The serotonin transporter belongs to the family of sodium-chloride coupled neurotransmitter transporter and is related to depression in humans. It is therefore an important drug target to support treatment of depression. Recently, structures of human serotonin transporter in complex with inhibitor molecules have been published.

An engineered monomer binding-protein for α-synuclein efficiently inhibits the proliferation of amyloid fibrils.

Removing or preventing the formation of [Formula: see text]-synuclein aggregates is a plausible strategy against Parkinson's disease. To this end, we have engineered the [Formula: see text]-wrapin AS69 to bind monomeric [Formula: see text]-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of [Formula: see text]-synuclein and formation of visible [Formula: see text]-synuclein aggregates.

Biallelic mutation of human encoding the taurine transporter TAUT is linked to early retinal degeneration.

We previously reported that inactivation of the transmembrane taurine transporter (TauT or solute carrier 6a6) causes early retinal degeneration in mice. Compatible with taurine's indispensability for cell volume homeostasis, protein stabilization, cytoprotection, antioxidation, and immuno- and neuromodulation, mice develop multisystemic dysfunctions (hearing loss; liver fibrosis; and behavioral, heart, and skeletal muscle abnormalities) later on. Here, by genetic, cell biologic, H-magnetic resonance spectroscopy and molecular dynamics simulation studies, we conducted in-depth characterization of a novel disorder: human TAUT deficiency.

Farnesylation of human guanylate-binding protein 1 as safety mechanism preventing structural rearrangements and uninduced dimerization.

Human guanylate-binding protein 1 (hGBP1) belongs to the family of dynamin-like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post-translationally modified by attachment of a farnesyl group yielding farn-hGBP1. In this study, hydrodynamic differences in farn-hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size-exclusion chromatography (SEC).

High-throughput stability screening for detergent-solubilized membrane proteins.

Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization.

Mechanism of Fibril and Soluble Oligomer Formation in Amyloid Beta and Hen Egg White Lysozyme Proteins.

Assembly and deposition of insoluble amyloid fibrils with a distinctive cross-β-sheet structure is the molecular hallmark of amyloidogenic diseases affecting the central nervous system as well as non-neuropathic amyloidosis. Amyloidogenic proteins form aggregates via kinetic pathways dictated by initial solution conditions. Often, early stage, cytotoxic, small globular amyloid oligomers (gOs) and curvilinear fibrils (CFs) precede the formation of late-stage rigid fibrils (RFs).

Interaction of carbohydrate-binding modules with poly(ethylene terephthalate).

Poly(ethylene terephthalate) (PET) is one of the most widely applied synthetic polymers, but its hydrophobicity is challenging for many industrial applications. Biotechnological modification of PET surface can be achieved by PET hydrolyzing cutinases. In order to increase the adsorption towards their unnatural substrate, the enzymes are fused to carbohydrate-binding modules (CBMs) leading to enhanced activity.

Pseudomonas aeruginosa esterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization.

The human membrane-bound α/β-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized.

α-Synuclein-derived lipoparticles in the study of α-Synuclein amyloid fibril formation.

Aggregation of the protein α-Synuclein (αSyn) is of great interest due to its involvement in the pathology of Parkinson's disease. However, under in vitro conditions αSyn is very soluble and kinetically stable for extended time periods. As a result, most αSyn aggregation assays rely on conditions that artificially induce or enhance aggregation, often by introducing rather non-native conditions.

Structural Studies of Autophagy-Related Proteins.

Information about the structure and dynamics of proteins is crucial for understanding their physiological functions as well as for the development of strategies to modulate these activities. In this chapter we will describe the work packages required to determine the three-dimensional structures of proteins involved in autophagy by using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Further we will provide instructions how to perform a molecular dynamics (MD) simulation using GABARAP as example protein.

Clustering cryo-EM images of helical protein polymers for helical reconstructions.

Helical protein polymers are often dynamic and complex assemblies, with many conformations and flexible domains possible within the helical assembly. During cryo-electron microscopy reconstruction, classification of the image data into homogeneous subsets is a critical step for achieving high resolution, resolving different conformations and elucidating functional mechanisms. Hence, methods aimed at improving the homogeneity of these datasets are becoming increasingly important.

Integrated NMR, Fluorescence, and Molecular Dynamics Benchmark Study of Protein Mechanics and Hydrodynamics.

Understanding the function of a protein requires not only knowledge of its tertiary structure but also an understanding of its conformational dynamics. Nuclear magnetic resonance (NMR) spectroscopy, polarization-resolved fluorescence spectroscopy and molecular dynamics (MD) simulations are powerful methods to provide detailed insight into protein dynamics on multiple time scales by monitoring global rotational diffusion and local flexibility (order parameters) that are sensitive to inter- and intramolecular interactions, respectively. We present an integrated approach where data from these techniques are analyzed and interpreted within a joint theoretical description of depolarization and diffusion, demonstrating their conceptual similarities.

N-Terminal Domains of Cardiac Myosin Binding Protein C Cooperatively Activate the Thin Filament.

Muscle contraction relies on interaction between myosin-based thick filaments and actin-based thin filaments. Myosin binding protein C (MyBP-C) is a key regulator of actomyosin interactions. Recent studies established that the N'-terminal domains (NTDs) of MyBP-C can either activate or inhibit thin filaments, but the mechanism of their collective action is poorly understood.

Origin of metastable oligomers and their effects on amyloid fibril self-assembly.

Assembly of rigid amyloid fibrils with their characteristic cross-β sheet structure is a molecular signature of numerous neurodegenerative and non-neuropathic disorders. Frequently large populations of small globular amyloid oligomers (gOs) and curvilinear fibrils (CFs) precede the formation of late-stage rigid fibrils (RFs), and have been implicated in amyloid toxicity. Yet our understanding of the origin of these metastable oligomers, their role as on-pathway precursors or off-pathway competitors, and their effects on the self-assembly of amyloid fibrils remains incomplete.

α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay.

Studying the aggregation of amyloid proteins like α-synuclein is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors (, aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive fluorescence reporter of amyloid fibril formation and are conducted in a plate-reader-based format, permitting the simultaneous screening of multiple samples and conditions. However, aggregation assays are generally prone to poor reproducibility due to the stochastic nature of fibril nucleation and the multiplicity of modulating factors.

Advancements of the sFIDA method for oligomer-based diagnostics of neurodegenerative diseases.

Early diagnosis of Alzheimer's disease (AD) is of great importance for the development of therapeutics and their application in the clinical environment. Amyloid β (Aβ) oligomers are crucial for the onset and progression of AD and represent a popular drug target, being presumably the most direct biomarker. Efforts to measure Aβ oligomers in body fluids are hampered by the low analyte concentration and presence of Aβ monomers.

Biophysical insights from a single chain camelid antibody directed against the Disrupted-in-Schizophrenia 1 protein.

Accumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis.

Chemically Stable Lipids for Membrane Protein Crystallization.

Lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs.

The Aβ oligomer eliminating D-enantiomeric peptide RD2 improves cognition without changing plaque pathology.

While amyloid-β protein (Aβ) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer's disease (AD), soluble oligomeric Aβ has been hypothesized to be responsible for synapse damage, neurodegeneration, learning, and memory deficits in AD. Here, we investigate the in vitro and in vivo efficacy of the D-enantiomeric peptide RD2, a rationally designed derivative of the previously described lead compound D3, which has been developed to efficiently eliminate toxic Aβ42 oligomers as a promising treatment strategy for AD. Besides the detailed in vitro characterization of RD2, we also report the results of a treatment study of APP/PS1 mice with RD2.

Pyroglutamate-modified Aβ(3-42) affects aggregation kinetics of Aβ(1-42) by accelerating primary and secondary pathways.

The aggregation into amyloid fibrils of amyloid-β (Aβ) peptides is a hallmark of Alzheimer's disease. A variety of Aβ peptides have been discovered , with pyroglutamate-modified Aβ (pEAβ) forming a significant proportion. pEAβ is mainly localized in the core of plaques, suggesting a possible role in inducing and facilitating Aβ oligomerization and accumulation.