Water-lipid interface in lipidic mesophases with excess water.

This study investigates the influence of excess water on the lipidic mesophase during the phase transition from diamond cubic phase (Pn3̄m) to reverse hexagonal phase (HII). Using a combination of small angle X-ray scattering (SAXS), broadband dielectric spectroscopy (BDS), and Fourier transform infrared (FTIR) techniques, we explore the dynamics of lipids and their interaction with water during phase transition. Our BDS results reveal three relaxation processes originating from lipids, all of which exhibit a kink during the phase transition. With the excess water, these processes accelerate due to the plasticizing effect of water. Additionally, our results demonstrate that the headgroups in the HII phase are more densely packed than those in the Pn3̄m phase, which agrees with the FTIR results. Meanwhile, we investigate the influence of excess water on the lipid headgroups, the H-bond network of water, the lipid tail, and the interface carbonyl group between the head and tail of the lipid molecule. The results indicate that excess water permeates the lipid interface and forms additional hydrogen bonds with the carbonyl groups. As a result, the headgroups are more flexible in a lipidic mesophase with excess water than those in mesophases without excess water.

Amyloidogenic and non-amyloidogenic molten globule conformation of ß-lactoglobulin in self-crowded regime.

Molecular insights on the ß-lactoglobulin thermal unfolding and aggregation are derived from FTIR and UV Resonance Raman (UVRR) investigations. We propose an in situ and in real-time approach that thanks to the identification of specific spectroscopic markers can distinguish the two different unfolding pathways pursued by ß-lactoglobulin during the conformational transition from the folded to the molten globule state, as triggered by the pH conditions. For both the investigated pH values (1.4 and 7.5) the greatest conformational variation of ß-lactoglobulin occurs at 80 °C and a high degree of structural reversibility after cooling is observed. In acidic condition ß-lactoglobulin exposes to the solvent its hydrophobic moieties in a much higher extent than in neutral solution, resulting on a highly open conformation. Moving from the diluted to the self-crowded regime, the solution pH and consequently the different molten globule conformation select the amyloid or non-amyloid aggregation pathway. At acidic condition the amyloid aggregates form during the heating cycle leading to the formation of transparent hydrogel. On the contrary, in neutral condition the amyloid aggregates never form. Information on the secondary structure conformational change of ß-lactoglobulin and the formation of amyloid aggregates are obtained by FTIR spectroscopy and are related to the information of the structural changes localized around the aromatic amino acid sites by UVRR technique. Our results highlight a strong involvement of the chain portions where tryptophan is located on the formation of amyloid aggregates.

Multi-length scale structural investigation of lysozyme self-assembly.

Reactive amyloid oligomers are responsible for cytotoxicity in amyloid pathologies and because of their unstable nature characterizing their behavior is a challenge. The physics governing the self-assembly of proteins in crowded conditions is extremely complex and its comprehension, despite its paramount relevance to understanding molecular mechanisms inside cells and optimizing pharmaceutical processes, remains inconclusive. Here, we focus on the amyloid oligomerization process in self-crowded lysozyme aqueous solutions in acidic conditions. We reveal that the amyloid oligomers form at high protein concentration and low pH. Through multi-length scale spectroscopic investigations, we find that amyloid oligomers can further interconnect with each other by weak and non-specific interactions forming an extended network that leads to the percolation of the whole system. Our multi-length scale structural analysis follows the thermal history of amyloid oligomers from different perspectives and highlights the impact of hierarchical self-assembly of biological macromolecules on functional properties.

Probing Water State during Lipidic Mesophases Phase Transitions.

We investigate the static and dynamic states of water network during the phase transitions from double gyroid ( Ia3‾d ) to double diamond ( Pn3‾m ) bicontinuous cubic phases and from the latter to the reverse hexagonal (HII ) phase in monolinolein based lipidic mesophases by combining FTIR and broadband dielectric spectroscopy (BDS). In both cubic(s) and HII phase, two dynamically different fractions of water are detected and attributed to bound and interstitial free water. The dynamics of the two water fractions are all slower than bulk water due to the hydrogen-bonds between water molecules and the lipid's polar headgroups and to nanoconfinement. Both FTIR and BDS results suggest that a larger fraction of water is hydrogen-bonded to the headgroup of lipids in the HII phase at higher temperature than in the cubic phase at lower temperature via H-bonds, which is different from the common expectation that the number of H-bonds should decrease with increase of temperature. These findings are rationalized by considering the topological ratio of interface/volume of the two mesophases.

Hydrogen Bonding and Solvation of a Proline-Based Peptide Model in Salt Solutions.

The hydrogen bonding of water and water/salt mixtures around the proline-based tripeptide model glycyl-l-prolyl-glycinamide·HCl (GPG-NH2) is investigated here by multi-wavelength UV resonance Raman spectroscopy (UVRR) to clarify the role of ion-peptide interactions in affecting the conformational stability of this peptide. The unique sensitivity and selectivity of the UVRR technique allow us to efficiently probe the hydrogen bond interaction between water molecules and proline residues in different solvation conditions, along with its influence on trans to cis isomerism in the hydrated tripeptide. The spectroscopic data suggest a relevant role played by the cations in altering the solvation shell at the carbonyl site of proline., while the fluoride and chloride anions were found to promote the establishment of the strongest interactions on the C=O site of proline. This latter effect is reflected in the greater stabilization of the trans conformers of the tripeptide in the presence of these specific ions. The molecular view provided by UVRR experiments was complemented by the results of circular dichroism (CD) measurements that show a strong structural stabilizing effect on the ß-turn motif of GPG-NH2 observed in the presence of KF as a co-solute.

Insight into the thermal stability of DNA in hydrated ionic liquids from multi-wavelength UV resonance Raman experiments.

The utility of ionic liquids (ILs) as alternative solvents for stabilizing and preserving the native structure of DNA over the long term may be envisaged for biotechnological and biomedical applications in the near future. The delicate balance between the stabilizing and destabilizing effects of IL-mediated interactions with the structure of DNA is complex and is still not well understood. This work reports a fundamental study dealing with the effect exerted by cations and anions in imidazolium-based ILs on the thermal structural stability of large nucleic acid molecules. Multi-wavelength UV resonance Raman spectroscopy is used for selectively detecting heat-induced structural transitions of DNA localized on specific base tracts. Our study reveals the establishment of preferential interactions between the imidazolium cations of ILs and the guanine bases in the DNA groove that lead to more effective stacking between the guanine bases even at high temperatures. Interestingly, we observe that this trend for ILs sharing the same chloride anion is further enhanced as the alkyl chain on the imidazolium cation gets shorter. The results from the present investigation lead to a more comprehensive view of the IL-mediated interactions with A-T and G-C base pairs during thermal unfolding.

Amyloid Self-Assembly of Lysozyme in Self-Crowded Conditions: The Formation of a Protein Oligomer Hydrogel.

A method is designed to quickly form protein hydrogels, based on the self-assembly of highly concentrated lysozyme solutions in acidic conditions. Their properties can be easily modulated by selecting the curing temperature. Molecular insights on the gelation pathway, derived by in situ FTIR spectroscopy, are related to calorimetric and rheological results, providing a consistent picture on structure-property correlations. In these self-crowded samples, the thermal unfolding induces the rapid formation of amyloid aggregates, leading to temperature-dependent quasi-stationary levels of antiparallel cross ß-sheet links, attributed to kinetically trapped oligomers. Upon subsequent cooling, thermoreversible hydrogels develop by the formation of interoligomer contacts. Through heating/cooling cycles, the starting solutions can be largely recovered back, due to oligomer-to-monomer dissociation and refolding. Overall, transparent protein hydrogels can be easily formed in self-crowding conditions and their properties explained, considering the formation of interconnected amyloid oligomers. This type of biomaterial might be relevant in different fields, along with analogous systems of a fibrillar nature more commonly considered.

Rigid, Fibrillar Quaternary Structures Induced by Divalent Ions in a Carboxylated Linear Polysaccharide.

Polysaccharides are ubiquitous in nature; they serve fundamental roles in vivo and are used for a multitude of food, pharmaceutical, cosmetic biomaterials, and biomedical applications. Here, the structure-property function for low acetylated Gellan gum hydrogels induced by divalent ions was established by means of optical, rheological, and microscopic techniques. The hydrogels interacted with visible light as revealed by birefringence and multiple scattering, as a consequence of quaternary, supramolecular fibrillar structures. The molecular assembly and structure were elucidated by statistical analysis and polymer physics concepts applied to high-resolution AFM height images and further supported by FTIR. This revealed intramolecular coil-to-single helix transitions, followed by lateral aggregation of single helices into rigid, fibrillar quaternary structures, ultimately responsible for gelation of the system. Calcium and magnesium chloride were shown to lead to fibrils up to heights of 6.0 nm and persistence lengths of several micrometers. The change in molecular structure affected the macroscopic gel stiffness, with the plateau shear modulus reaching ∼105 Pa. These results shed light on the two-step gelation mechanism of linear polysaccharides, their conformational molecular changes at the single polymer level and ultimately the macroscale properties of the ensued gels.