  Enhanced stabilisation of protein structures via the presence of inert
excipients is a key mechanism adopted both by physiological systems and in
biotechnological applications. While the intrinsic stability of proteins is
ultimately fixed by their amino acid composition and organisation, the
interactions between excipients and proteins together with their concentrations
introduce an additional layer of complexity and in turn, method of modulating
protein stability. Here, we combined experimental measurements with molecular
dynamics simulations and graph-theory based analyses to assess the
stabilising/destabilising effects of different kinds of osmolytes on proteins
during heat-mediated denaturation. We found that (i) proteins in solution with
stability-enhancing osmolytes tend to have more compact interaction networks
than those assumed in presence of destabilising excipients; (ii) a strong
negative correlation (R = -0.85) characterises the relationship between the
melting temperature Tm and the preferential interaction coefficient defined by
the radial distribution functions of osmolytes and water around the protein and
(iii) a positive correlation exists between osmolyte-osmolyte clustering and
the extent of preferential exclusion from the local domain of the protein,
suggesting that exclusion may be driven by enhanced steric hindrance of
aggregated osmolytes.
