Solvation water role in driving structural conformation and self-assembly of peptides and proteins

“Solvation water role in driving structural conformation and self-assembly of peptides and proteins”, the title of the present thesis, summarizes the objective of my three years PhD course aimed to investigate the relationship existing between the structure of biomolecules in solution and their mutual influence with surrounding water molecules. The three year work has been mainly experimental and principally focused in analyzing the capability of vibrational spectroscopies and some non-linear spectroscopic techniques to disentangle various contributions to solvent-molecule interactions. However to gain a deeper insight in the comprehension of such a complex problem, different types of experimental methods and theoretical modeling have supplemented spectroscopic techniques. A general survey of UV Resonant Raman (UVRR) spectroscopy and picosecond transient grating (TG) technique is reported in Chapter 2. The specific interactions of water molecules with glutathione tripeptide dissolved in pure water and water/salts mixtures are investigated experimentally by UVRR spectroscopy and modeled by molecular dynamics simulations. The results are presented in Chapter 3 and focus on the peptide-solvent interactions at the peptide site of glutathione thanks to the high selectivity of UVRR spectroscopy. Spectra are collected and analyzed as a function of concentration, pH, temperature and ion nature. OH stretching and amide modes spectral regions result very sensitive to the variations of the experimental conditions. The output provides a picture of the hydrogen-bonding network around glutathione. The number and the strength of hydrogen bonds increase in the deprotonated form of the tripeptide that exhibits a more marked capacity in decreasing the intermolecular order of water in its hydration shell. Potassium salts and imidazolium based ionic liquids of the halogen series are used to investigate the ions effect on glutathione structure and hydration shell. UVRR spectra present specific features possessing a great dependence on the nature of the anion present in solution rather than that of the cation, suggesting a strong capacity of anions to modify the glutathione structure and its hydration shell. There is a strong evidence that chloride and bromide anions interact at the NH site of glutathione reducing the possibility to form hydrogen bonds with water molecules and making the environment more hydrophobic than in pure water. Instead, iodide anion increases the number of water molecules at the peptide site, creating a strong polar environment. The spectroscopic and computational data are in perfect agreement and their interpretation can be based on the peptide link resonance model. In all the studied solvation environments, a progressive reduction in the strength of hydrogen bond interactions on amide sites is probed upon the increment of temperature, accompanied by conformational changes involving also the trans-cis isomerization of glutathione. Chapter 4 deals with the results of the study on self-crowded lysozyme solutions characterized by different degrees of aggregation and networking. Lysozyme has been widely investigated, as a convenient model protein, due to its ability to form amyloid fibrils in acidic conditions at high temperatures. Most of these past studies involved rather diluted samples in which fibril assembly is relatively slow. More rarely the formation of amyloid aggregates was examined in concentrated conditions, despite their relevance in different fields, from cellular biology and medicine to biomaterial and food technologies. In the present study, thermal unfolding and aggregation of highly concentrated (>100 mg/ml) lysozyme solutions at pH=1.8 are investigated. A method is designed to form protein hydrogels in a few hours. Their properties can be easily modulated selecting the curing temperature. The whole gelation process was monitored in situ by Fourier transform infrared (FTIR) spectroscopy assisted by hydrogen/deuterium isotopic exchange, to probe conformational changes and amyloid structuring. Specific molecular conformations are put in relation to thermodynamic properties by calorimetric measurements, to structural information by small angle x-ray and neutron scattering and to viscoelastic properties by means of rheology and TG experiments. This multi-technique approach is necessary in order to obtain a consistent picture on structure-property correlation in self-crowded protein samples. Aggregates constituted by antiparallel cross β-sheet links grow up quickly (less than two hours) within the 45-60 °C temperature range, leading to temperature-dependent quasi-stationary level of amyloid structures, attributed to kinetically trapped oligomers. Upon subsequent cooling, hydrogels develop quickly through the formation of non-specific inter-oligomer contacts. Due to this supramolecular assembly, the hydrogel is transparent, thermo-reversible and rather weak from a mechanical point of view. Lysozyme solutions can be recovered back to a large extent, following a process of oligomer-to-monomer dissociation and refolding. Overall, evidence is given of the possibility of easily forming protein hydrogels in self-crowding conditions constituted by kinetically trapped amyloid oligomers, interconnected by weak interactions. This type of gels might be relevant in different fields, when concentrated protein systems experience denaturing conditions.