FTIR analysis of the high pressure response of native insulin assemblies

It is widely recognized that a central role in conferring stability to the structure of proteins against misfolding and aggregation is played by the formation of oligomers. The case of insulin is prototypical in this respect: in our body it is stored up in stable inactive hexameric assemblies whereas only in its monomeric form it recovers the role of regulating carbohydrate and fat metabolism. In the present paper, exploiting the optimal coupling between FTIR spectroscopy and diamond anvil cell technique, we probe the stability of different insulin oligomeric forms under high pressure, namely over the ranges 0-15 kbar for water solution and 0-80 kbar for dry powder. Results obtained show di…

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 Parkinson's 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 fragmentati…

Raman analysis of insulin denaturation induced by high-pressure and thermal treatments

Raman spectroscopy has been used to investigate different conformational states of bovine pancreatic insulin: the native form and several structurally modified states with different extent of denaturation induced by thermo-chemical treatment and by applying very high pressure (up to 8 GPa) using a diamond anvil cell. High-pressure results confirm the peculiar strength to volume compression of insulin and largely extend the pressure range of its structural stability (0-4.2 GPa). Above 4.2 GPa, insulin undergoes an irreversible structural transition that, once pressure is released, leaves the sample in a new conformational state. The protein secondary structure after the pressure treatment re…

Nouvelles perspectives concernant la structure et la fonction du domaine carboxyl terminal de Hfq

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…

Decoding vibrational states of Concanavalin A amyloid fibrils.

International audience; 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 deta…

THz spectroscopy studies on proteins: exploring collective modes of amyloid fibrils

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 Alzheimer's and Parkinson's 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/…

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 par…

Conformational Transitions upon Maturation Rule Surface and pH-Responsiveness of α-Lactalbumin Microparticulates

De novo designed protein supramolecular structures are nowadays attracting much interest as highly performing biomaterials. While a clear advantage is provided by the intrinsic biocompatibility and...

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 Parkinson's 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-sh…

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

High Pressure (HP) Fourier Transform Infrared Spectroscopy (FTIR) 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 few hours; after this time the infrared absorption of fibrils becomes stable. It is here shown how the 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, …

Infrared microspectroscopy of biochemical response of living cells in microfabricated devices

Abstract First experiments demonstrating the suitability of novel microfabricated fluidic devices for measuring living cells in physiological environment by synchrotron radiation (SR) Fourier Transform Infrared microspectroscopy (μ-FTIR) are presented. The devices were fabricated on CaF 2 windows, using the photoresist XARP 3100/10 to define the liquid cell lay-out. Therefore, the sample holder is transparent to both visible and infrared light, robust, completely recyclable and with a precise spacing. Using prototype devices of thicknesses 9, 5 and 3 μm, we studied the response of the U937 monocytic cell line to mechanical compression. The temporal evolution of the FTIR spectra, characteris…

Aggregation properties of proteins under high pressure

Fourier transform infrared spectroscopy (FTIR) coupled with High Pressure (HP) techniques is a suitable tool to investigate unfolding/misfolding processes providing useful information on the kinetics of aggregation of proteins[1]. Since HP affects only the volume contribution to the Gibbs free energy, it is able to perturb the structure of proteins in a reversible way [2][3]. The principle governing pressure effects is that it tends to shift a system towards the state that occupies the smallest volume, it causes the electrostriction of charged and polar groups, the elimination of packing defects, and the solvation of hydrophobic groups. Cavities and packing defects are expected to be major …

Terahertz Absorption change in Photosynthetic Reaction Center upon photoactivation

Photosynthetic reaction center is a membrane protein which plays an important role in converting solar energy into chemical energy. Upon continuous illumination a charge separation occurs within the protein which is stable for several seconds [1]. The stability of this state is very important to protect the protein against the release of excess heat and an instable radical due to charge recombination. Absorption of terahertz radiation corresponds to the excitation of large vibrations in the protein and is thus suitable for studying its different dynamical states. Here we recorded terahertz absorption spectra of the dark and illuminated state of reaction centre from the purple membrane bacte…