Structural Model for an Alkaline Form of Ferricytochrome c

An ^(15)N-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy. The mutations were planned to produce a cytochrome c with a single conformer. Despite suboptimal conditions for the collection of spectra (i.e., pH ≈ 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism. 76% of amino acids and 49% of protons were assigned successfully. The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions. Lys73 was assigned as the axial ligand together with His18. In this manner, 838 meaningful NOEs for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5 ± 0.2 Å. Most of the native cytochrome c conformation is maintained at high pH. The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact. Structural differences are concentrated in the 70−80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent. Based on several spectral features, we conclude that the folded polypeptide is highly fluxional

Alpha-Synuclein-Nanoparticle Interactions: Understanding, Controlling and Exploiting Conformational Plasticity

Alpha-synuclein (alpha S) is an extensively studied protein due to its involvement in a group of neurodegenerative disorders, including Parkinson ' s disease, and its documented ability to undergo aberrant self-aggregation resulting in the formation of amyloid-like fibrils. In dilute solution, the protein is intrinsically disordered but can adopt multiple alternative conformations under given conditions, such as upon adsorption to nanoscale surfaces. The study of alpha S-nanoparticle interactions allows us to better understand the behavior of the protein and provides the basis for developing systems capable of mitigating the formation of toxic aggregates as well as for designing hybrid nanomaterials with novel functionalities for applications in various research areas. In this review, we summarize current progress on alpha S-nanoparticle interactions with an emphasis on the conformational plasticity of the biomolecule

A facile Oxygen-17 NMR Method to Determine Effective Viscosity in Dilute, Molecularly Crowded and Confined Aqueous Media

This NMR probe of water dynamics enables viscosity determination in concentrated and crowded solutions and allows quantifying internal fluidity within biological condensates

Espresso Coffee Mitigates the Aggregation and Condensation of Alzheimer′s Associated Tau Protein

: Espresso coffee is among the most consumed beverages in the world. Recent studies report a protective activity of the coffee beverage against neurodegenerative disorders such as Alzheimer's disease. Alzheimer's disease belongs to a group of disorders, called tauopathies, which are characterized by the intraneuronal accumulation of the microtubule-associated protein tau in fibrillar aggregates. In this work, we characterized by NMR the molecular composition of the espresso coffee extract and identified its main components. We then demonstrated with in vitro and in cell experiments that the whole coffee extract, caffeine, and genistein have biological properties in preventing aggregation, condensation, and seeding activity of the repeat region of tau. We also identified a set of coffee compounds capable of binding to preformed tau fibrils. These results add insights into the neuroprotective potential of espresso coffee and suggest candidate molecular scaffolds for designing therapies targeting monomeric or fibrillized forms of tau

Conformational signatures induced by ubiquitin modification in the amyloid-forming tau repeat domain

Posttranslational modifications can critically affect conformational changes of amyloid-forming proteins. Ubiquitination of the microtubule-associated tau protein, an intrinsically disordered biomolecule, has been proposed to influence the formation of filamentous deposits in neurodegenerative conditions. Given the reported link between aggregation propensity and intrinsic structural preferences (e.g., transient extended structural motifs or tertiary contacts) in disordered proteins, we sought to explore the conformational landscape of ubiquitinated tau. Exploiting selective conjugation reactions, we produced single- and double-monoubiquitinated protein samples. Next, we examined the ubiquitinated species from different standpoints using NMR spectroscopy, small-angle X-ray scattering experiments, and native ion mobility–mass spectrometry (IM–MS). Moreover, we obtained atomistic representations of the conformational ensembles via scaled MD calculations, consistent with the experimental data. Modifying the repeat domain of tau with ubiquitin had a limited effect on secondary structure propensities and local mobility of distal regions. Instead, ubiquitination enhanced the compaction of the conformational ensemble, with the effect modulated by the site and the number of modifications. Native IM–MS patterns pinpointed similarities and differences between distinct tau proteoforms. It emerges that ubiquitination exerts a position-specific influence on the conformational distribution of tau molecules. This study reveals the unique conformational features of ubiquitinated forms of tau and points to their potential impact on aggregation and phase separation propensities, offering clues for a better understanding of disease-related structural alterations

Conformational signatures induced by ubiquitin modification in the amyloid-forming tau repeat domain

Posttranslational modifications can critically affect conformational changes of amyloid-forming proteins. Ubiquitination of the microtubule-associated tau protein, an intrinsically disordered biomolecule, has been proposed to influence the formation of filamentous deposits in neurodegenerative conditions. Given the reported link between aggregation propensity and intrinsic structural preferences (e.g., transient extended structural motifs or tertiary contacts) in disordered proteins, we sought to explore the conformational landscape of ubiquitinated tau. Exploiting selective conjugation reactions, we produced single- and double-monoubiquitinated protein samples. Next, we examined the ubiquitinated species from different standpoints using NMR spectroscopy, small-angle X-ray scattering experiments, and native ion mobility–mass spectrometry (IM–MS). Moreover, we obtained atomistic representations of the conformational ensembles via scaled MD calculations, consistent with the experimental data. Modifying the repeat domain of tau with ubiquitin had a limited effect on secondary structure propensities and local mobility of distal regions. Instead, ubiquitination enhanced the compaction of the conformational ensemble, with the effect modulated by the site and the number of modifications. Native IM–MS patterns pinpointed similarities and differences between distinct tau proteoforms. It emerges that ubiquitination exerts a position-specific influence on the conformational distribution of tau molecules. This study reveals the unique conformational features of ubiquitinated forms of tau and points to their potential impact on aggregation and phase separation propensities, offering clues for a better understanding of disease-related structural alterations

Stable ubiquitin conjugation for biological interrogation of ubiquitinated tau repeat domain

Protein semisynthesis approaches are key for gaining insights into the effects of post-translational modifications (PTMs) on the structure and function of modified proteins. Among PTMs, ubiquitination involves the conjugation of a small protein modifier to a substrate amino acid residue and is unique in controlling a variety of cellular processes. Interest has grown in understanding the role of ubiquitination in neurodegenerative conditions, including tauopathies. The latter are characterized by the accumulation of the intrinsically disordered protein tau in the form of neurofibrillary tangles in the brains of patients. The presence of ubiquitinated tau in the pathological aggregates suggests that ubiquitination might play a role in the formation of abnormal protein deposits. In this study, we developed a new strategy, based on dehydroalanine chemistry, to install wild type ubiquitin on a tau repeat domain construct with site-specificity. We optimized a three-step reaction which yielded a good amount of highly pure tau repeat domain ubiquitinated in position 353. The structural features of the conjugate were examined by circular dichroism and NMR spectroscopy. The ubiquitinated tau was challenged in a number of assays: fibrils formation under aggregating conditions in vitro, chemical stability upon exposure to a variety of biological media including cell extracts, and internalization into astrocytes. The results demonstrated the wide applicability of the new semisynthetic strategy for the investigation of ubiquitinated substrates in vitro or in cell, and in particular for studying if ubiquitination has a role in the molecular mechanisms that underlie the aberrant transition of tau into pathological aggregates

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters

Are they in or out? The elusive interaction between Qtracker(\uae)800 vascular labels and brain endothelial cells

AIM:Qtracker\uae800 Vascular labels (Qtracker\uae800) are promising biomedical tools for high-resolution vasculature imaging; their effects on mouse and human endothelia, however, are still unknown.MATERIALS & METHODS:Qtracker\uae800 were injected in Balb/c mice, and brain endothelium uptake was investigated by transmission electron microscopy 3-h post injection. We then investigated, in vitro, the effects of Qtracker\uae800 exposure on mouse and human endothelial cells by calcium imaging.RESULTS:Transmission electron microscopy images showed nanoparticle accumulation in mouse brain endothelia. A subset of mouse and human endothelial cells generated intracellular calcium transients in response to Qtracker\uae800.CONCLUSION:Qtracker\uae800 nanoparticles elicit endothelial functional responses, which prompts biomedical safety evaluations and may bias the interpretation of experimental studies involving vascular imaging