• proteins
• unfolding
• glass transition
• fast calorimetry
• neutron scattering

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.