Fast dynamics and melting process of globular proteins embedded in glass-forming solvents

This thesis project is focused on experimental studies of dynamics and thermodynamic transitions of biomacromolecules embedded in non aqueous glass-forming systems. The main effort was to investigate such systems by a novel experimental technique, i.e. fast differential scanning calorimetry (FDSC). FDSC is a particular kind of differential calorimetry, also called chip calorimetry, that is based on a thin film of sample deposited on MEMS (microelectro-mechanical-system) calorimetric devices, instead of the usual sample holded in metallic pans in furnaces as for conventional calorimetry. This switch of paradigm leads to the reduction of the involved sample mass and allows one to reach very high heating scan rates, up to 40000 K/s, without loosing precision nor being affected by significant thermal lags or gradients. The consequent improvement (by more than three decades) in the available heating rates compared to conventional calorimetry provides new opportunities for a more detailed investigation of metastable materials, along with a stronger suppression of diffusive and reorganization processes. It also allows for a better insight in the investigation of kinetic processes down to millisecond timescales. In this thesis the class of systems studied so far by FDSC was further expanded by choosing protein solutions in non aqueous glass-forming solvents, that are materials of interest for cryo- and dry preservation. For the first time the whole temperature interval ranging from the glassy state up to the unfolding high temperature region has been studied by FDSC. Such a challenge has required the development of new ad-hoc methods of analysis and a thorough comparison with those employed in literature. As preliminary study, the new methods have been tested on four prototypical glass forming systems, namely glycerol, glucose, sucrose and orto-terphenyl, for which the rate dependence of glass transition and the enthalpy recovery kinetics were estimated. Once defined a suitable operative method, the work moved to investigate lysozyme, a very well-known globular protein, often used as a benchmark. The choice of lysozyme was supported by very robust and in-depth characterization available in literature, enabling an effective comparison with here achieved results. Glycerol was used as solvent, since it is known to not significatively alter the three-dimensional structure of the biomacromolecule while it enhances its thermostability. Moreover, it is often used as a cryoprotectant and as a solvent/eccipient for drugs. Finally, its low vapour pressure and high boiling point allow a wide temperature range to be investigated for solutions. Solutions at three concentrations have been studied to bring out the mutual effects of the two components of the mixture, related to the proteinsolvent interaction. Both the glass transition as well as the unfolding phenomenon have been studied in these solutions, at low and high temperatures, respectively. The wide range of temperature scan rates enabled by FDSC permitted an accurate estimate of the dynamics involved in glass transition. At high temperature an endothermic transition, known also as melting, was found under heating: it is related to the unfolding mechanism through which a transition from the native to the denatured state of protein occurs. The calorimetric traces were found to be apparently irreversible and scan-rate dependent. The shape of the thermograms, as well as their dependence on scan rate, were employed to test some kinetic models for melting proposed in literature. For the two diluted solutions isothermal refolding experiments were also performed, shining a light on the complex mechanism of folding. Additionally, FDSC was applied to a solution of glycerol with thermolysin, a thermophilic enzyme that is more heat-resistant than lysozyme. The differences between the two proteins were so highlighted, in particular those in their physical behavior as affected by glycerol. Actually, the role of the solvent in controlling protein stablity and internal sub-nanosecond timescale motions has been recently demonstrated for lysozyme and similar mesophilic enzymes: fast fluctuations of proteins are affected by the viscosity of the surrounding solvent and, since they guide conformational changes, the thermal unfolding transition occurs at higher temperatures for more viscous solvents. Surprisingly, the amplitude of sub-nanosecond fluctuations reaches a common critical value for different solvents in proximity of the melting temperature, in analogy with the Lindemann criterion for the melting of solids. Such relation was here tested, on the thermophilic enzyme thermolysin embedded in three different deuterated solvents (water, glycerol and glucose): by means of elastic incoherent neutron scattering the temperature variation of the mean squared displacements of the hydrogen atoms spread all over the chain was catched and compared to the calorimetric melting temperatures. A criterion for thermal denaturation, based on a threshold for the mean squared displacement identifying the unfolding of the protein, was determined