Many research topics in the fields of condensed matter and the life sciences are based on small-angle X-ray and neutron scattering techniques. With the current rapid progress in source brilliance and detector technology, high data fluxes of ever-increasing quality are produced. In order to exploit such a huge quantity of data and richness of information, wider and more sophisticated approaches to data analysis are needed. Presented here isGENFIT, a new software tool able to fit small-angle scattering data of randomly oriented macromolecular or nanosized systems according to a wide list of models, including form and structure factors. Batches of curves can be analysed simultaneously in terms of common fitting parameters or by expressing the model parametersviaphysical or phenomenological link functions. The models can also be combined, enabling the user to describe complex heterogeneous systems.

Small-angle X-ray scattering (SAXS) and elastic and quasi-elastic neutron scattering techniques were used to investigate the high-pressure-induced changes on interactions, the low-resolution structure and the dynamics of lysozyme in solution. SAXS data, analysed using a global-fit procedure based on a new approach for hydrated protein form factor description, indicate that lysozyme completely maintains its globular structure up to 1500 bar, but significant modifications in the protein–protein interaction potential occur at approximately 600–1000 bar. Moreover, the mass density of the protein hydration water shows a clear discontinuity within this pressure range. Neutron scattering experiments indicate that the global and the local lysozyme dynamics change at a similar threshold pressure. A clear evolution of the internal protein dynamics from diffusing to more localized motions has also been probed. Protein structure and dynamics results have then been discussed in the context of protein–water interface and hydration water dynamics. According to SAXS results, the new configuration of water in the first hydration layer induced by pressure is suggested to be at the origin of the observed local mobility changes.

The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease M^proto cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the M^promonomer-dimer equilibrium dissociation constant. Since the functional unit of M^prois a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit Small Angle X-ray Scattering (SAXS) to investigate the structural features of SARS-CoV-2 M^proin solution as a function of protein concentration and temperature. A detailed thermodynamic picture of the monomer-dimer equilibrium is derived, together with the temperature-dependent value of the dissociation constant. SAXS is also used to study how the M^prodissociation process is affected by small inhibitors selected by virtual screening. We find that these inhibitors affect dimerization and enzymatic activity to a different extent and sometimes in an opposite way, likely due to the different molecular mechanisms underlying the two processes. The M^proresidues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed.

In solution small-angle neutron scattering has been used to study the solvation properties of lysozyme dissolved in water/glycerol mixtures. To detect the characteristics of the protein-solvent interface, 35 different experimental conditions (i.e., protein concentration, water/glycerol fraction in the solvent, content of deuterated compounds) have been considered and a suitable software has been developed to fit simultaneously the whole set of scattering data. The average composition of the solvent in the close vicinity of the protein surface at each experimental condition has been derived. In all the investigated conditions, glycerol resulted especially excluded from the protein surface, confirming that lysozyme is preferentially hydrated. By considering a thermodynamic hydration model based on an equilibrium exchange between water and glycerol from the solvation layer to the bulk, the preferential binding coefficient and the excess solvation number have been estimated. Results were compared with data previously derived for ribonuclease A in the same mixed solvent: even if the investigated solvent compositions were very different, the agreement between data is noticeable, suggesting that a unique mechanism presides over the preferential hydration process. Moreover, the curve describing the excess solvation number as a function of the solvent composition shows the occurrence of a region of maximal hydration, which probably accounts for the changes in protein stability detected in the presence of cosolvents.