Federica Piccirilli

New insight into the structure and function of Hfq C-terminus.

Accumulating evidence indicates that RNA metabolism components assemble into supramolecular cellular structures to mediate functional compartmentalization within the cytoplasmic membrane of the bacterial cell. This cellular compartmentalization could play important roles in the processes of RNA degradation and maturation. These components include Hfq, the RNA chaperone protein, which is involved in the post-transcriptional control of protein synthesis mainly by the virtue of its interactions with several small regulatory ncRNAs (sRNA). The Escherichia coli Hfq is structurally organized into two domains. An N-terminal domain that folds as strongly bent β-sheets within individual protomers to assemble into a typical toroidal hexameric ring. A C-terminal flexible domain that encompasses approximately one-third of the protein seems intrinsically unstructured. RNA-binding function of Hfq mainly lies within its N-terminal core, whereas the function of the flexible domain remains controversial and largely unknown. In the present study, we demonstrate that the Hfq-C-terminal region (CTR) has an intrinsic property to self-assemble into long amyloid-like fibrillar structures in vitro. We show that normal localization of Hfq within membrane-associated coiled structures in vivo requires this C-terminal domain. This finding establishes for the first time a function for the hitherto puzzling CTR, with a plausible central role in RNA transactions.

Sequential dissociation of insulin amyloids probed by high pressure Fourier transform infrared spectroscopy

High pressure (HP) Fourier transform infrared (FTIR) spectroscopy has been here employed to investigate the thermodynamic stability of bovine pancreatic insulin (BPI) amyloids. Once the aggregation reaction has started, the backbone arrangement of the proteins forming the amyloid is known to reach a stationary phase in a few hours; after this time the infrared absorption of fibrils becomes stable. It is here shown how further stabilization of the structure during the stationary phase can be probed via FTIR spectroscopy, through the observation of the high pressure behaviour of fibrils formed at different maturation stages. We report on the high pressure fragmentation of insulin amyloids, probed on fibrils formed in the early stages of the stationary phase. Moreover, we noticed a sequentiality high pressure dissociation that seems to respect a pre-existing hierarchy of structures: the stabilization of a protofibrillar state is observed at pressures in the order of a few kbar and our results suggest the possible occurrence of a partial refolding, induced by pressures up to 11.4 kbar. Our findings remark the importance of high pressure in stabilizing intermediate structures and in evaluating the driving forces of fibrillation, demonstrating how the control of electrostatic interactions and hydrophobic effects can be used to characterize the factors that modulate amyloids stability.

Decoding vibrational states of Concanavalin A amyloid fibrils.

Amyloid and amyloid-like fibrils are a general class of protein aggregates and represent a central topic in life sciences for their involvement in several neurodegenerative disorders and their unique mechanical and supramolecular morphological properties. Both their biological role and their physical properties, including their high mechanical stability and thermodynamic inertia, are related to the structural arrangement of proteins in the aggregates at molecular level. Significant variations may exist in the supramolecular organization of the commonly termed cross-β structure that constitutes the amyloid core. In this context, a fine knowledge of the structural details in fibrils may give significant information on the assembly process and on possible ways of tuning or inhibiting it. Here we propose a simple method based on the combined use of Fourier transform infrared spectroscopy and Fourier transform Raman spectroscopy to accurately reveal structural details in the fibrillar aggregates, side-chain exposure and intermolecular interactions. Interestingly, coupled analysis of mid-infrared spectra reveals antiparallel β-sheet orientation in ConA fibrils. We also report the comparison between THz absorption spectra of Concanavalin A in its native and fibrillar state at different hydration levels, allowing obtaining corroboration of peaks assignation in this range and information on the effect of amyloid supramolecular arrangement on the network dynamics of hydration water.

Infrared Microspectroscopy study of insulin crystals at high pressure

During the last years the coupling of high pressure techniques and infrared spectroscopy has proven to be a very powerful tool in the study of conformational changes of proteins. Protein unfolding and monomerization are events that are expected to take place at high pressure due to the peculiarity of pressure to shift the system towards the state that occupies the minimum volume. We observed the growth of apparently cubic crystals at a pressure of about 4 kbar, subjecting to high pressure a solution of misfolded insulin. Even if high pressure is commonly used to tune the growth rate of crystals, protein crystallization at high pressure is not a well known process and no evidences of the particular case of insulin are present in literature.

TeraFERMI: A Superradiant Beamline for THz Nonlinear Studies at the FERMI Free Electron Laser Facility

During the last few decades, we have witnessed an impressive advancement in the progress of THz technologies. THz spectroscopy can now be considered a mature technique, providing a formidable tool ...

Terahertz absorption of illuminated photosynthetic reaction center solution: a signature of photoactivation?

Photosynthetic reaction centers develop a stable charge separated state upon illumination. To investigate the molecular vibrations associated with the illuminated state of a reaction center we recorded terahertz absorption spectra of the photosynthetic reaction center from Rhodobacter sphaeroides in the dark and upon illumination and observed a small, but significant THz absorption increase in the 20 to 130 cm−1 spectral region. Reaction centers show very similar terahertz absorption increase when solubilized in detergents and in a lipidic sponge phase indicating that the nature of the bulk solvent has limited influence on the vibrational spectrum. The absorption change of the isolated LM subunit is very similar to that of the intact reaction center. Through temperature control experiments we show that 89% of the absorption change is likely attributed to the non-thermal activation of the protein molecules. These results indicate that picosecond molecular vibrations change primarily in the cofactors and/or in the evolutionary conserved core of the reaction center upon illumination, whereas the nuclear motions of the H-subunit and the bulk solvent have limited impact on the terahertz spectral changes.

High-Pressure-Driven Reversible Dissociation of α-Synuclein Fibrils Reveals Structural Hierarchy

The analysis of the α-synuclein (aS) aggregation process, which is involved in Parkinsons disease etiopathogenesis, and of the structural feature of the resulting amyloid fibrils may shed light on the relationship between the structure of aS aggregates and their toxicity. This may be considered a paradigm of the ground work needed to tackle the molecular basis of all the protein-aggregation-related diseases. With this aim, we used chemical and physical dissociation methods to explore the structural organization of wild-type aS fibrils. High pressure (in the kbar range) and alkaline pH were used to disassemble fibrils to collect information on the hierarchic pathway by which distinct β-sheets sequentially unfold using the unique possibility offered by high-pressure Fourier transform infrared spectroscopy. The results point toward the formation of kinetic traps in the energy landscape of aS fibril disassembly and the presence of transient partially folded species during the process. Since we found that the dissociation of wild-type aS fibrils by high pressure is reversible upon pressure release, the disassembled molecules likely retain structural information that favors fibril reformation. To deconstruct the role of the different regions of aS sequence in this process, we measured the high-pressure dissociation of amyloids formed by covalent chimeric dimers of aS (syn-syn) and by the aS deletion mutant that lacks the C-terminus, i.e., aS (1-99). The results allowed us to single out the role of dimerization and that of the C-terminus in the complete maturation of fibrillar aS.

Pressure effects on α-synuclein amyloid fibrils: An experimental investigation on their dissociation and reversible nature

α-synuclein amyloid fibrils are found in surviving neurons of Parkinsons disease affected patients, but the role they play in the disease development is still under debate. A growing number of evidences points to soluble oligomers as the major cytotoxic species, while insoluble fibrillar aggregates could even play a protection role. In this work, we investigate α-synuclein fibrils dissociation induced at high pressure by means of Small Angle X-ray Scattering and Fourier Transform Infrared Spectroscopy. Fibrils were produced from wild type α-synuclein and two familial mutants, A30P and A53T. Our results enlighten the different reversible nature of α-synuclein fibrils fragmentation at high pressure and suggest water excluded volumes presence in the fibrils core. Wild type and A30P species stabilized at high pressure are highly amyloidogenic and quickly re-associate into fibrils upon decompression, while A53T species shows a partial reversibility of the process likely due to the presence of an intermediate oligomeric state stabilized at high pressure. The amyloid fibrils dissociation process is here suggested to be associated to a negative activation volume, supporting the notion that α-synuclein fibrils are in a high-volume and high-compressibility state and hinting at the presence of a hydration-mediated activated state from which dissociation occurs.

Coherent THz Emission Enhanced by Coherent Synchrotron Radiation Wakefield

We demonstrate that emission of coherent transition radiation by a ∼1 GeV energy-electron beam passing through an Al foil is enhanced in intensity and extended in frequency spectral range, by the energy correlation established along the beam by coherent synchrotron radiation wakefield, in the presence of a proper electron optics in the beam delivery system. Analytical and numerical models, based on experimental electron beam parameters collected at the FERMI free electron laser (FEL), predict transition radiation with two intensity peaks at ∼0.3 THz and ∼1.5 THz, and extending up to 8.5 THz with intensity above 20 dB w.r.t. the main peak. Up to 80-µJ pulse energy integrated over the full bandwidth is expected at the source, and in agreement with experimental pulse energy measurements. By virtue of its implementation in an FEL beam dump line, this work promises dissemination of user-oriented multi-THz beamlines parasitic and self-synchronized to EUV and x-ray FELs.

Ethanol Controls the Self-Assembly and Mesoscopic Properties of Human Insulin Amyloid Spherulites

Protein self-assembly into amyloid fibrils or highly hierarchical superstructures is closely linked to neurodegenerative pathologies as Alzheimers and Parkinsons diseases. Moreover, protein assemblies also emerged as building blocks for bioinspired nanostructured materials. In both the above mentioned fields, the main challenge is to control the growth and properties of the final protein structure. This relies on a more fundamental understanding of how interactions between proteins can determine structures and functions of biomolecular aggregates. Here, we identify a striking effect of the hydration of the single human insulin molecule and solvent properties in controlling hydrophobicity/hydrophilicity, structures, and morphologies of a superstructure named spherulite, observed in connection to Alzheimers disease. Depending on the presence of ethanol, such structures can incorporate fluorescent molecules with different physicochemical features and span a range of mechanical properties and morphologies. A theoretical model providing a thorough comprehension of the experimental data is developed, highlighting a direct connection between the intimate physical protein-protein interactions, the growth, and the properties of the self-assembled superstructures. Our findings indicate structural variability as a general property for amyloid-like aggregates and not limited to fibrils. This knowledge is pivotal not only for developing effective strategies against pathological amyloids but also for providing a platform to design highly tunable biomaterials, alternative to elongated protein fibrils.