An experimentally representative in-silico protocol for dynamical studies of lyophilised and weakly hydrated amorphous proteins

Characterization of biopolymers in both dry and weakly hydrated amorphous states has implications for the pharmaceutical industry since it provides understanding of the effect of lyophilisation on stability and biological activity. Atomistic Molecular Dynamics (MD) simulations probe structural and dynamical features related to system functionality. However, while simulations in homogenous aqueous environments are routine, dehydrated model assemblies are a challenge with systems investigated in-silico needing careful consideration; simulated systems potentially differing markedly despite seemingly negligible changes in procedure. Here we propose an in-silico protocol to model proteins in lyophilised and weakly hydrated amorphous states that is both more experimentally representative and routinely applicable. Since the outputs from MD align directly with those accessed by neutron scattering, the efficacy of the simulation protocol proposed is shown by validating against experimental neutron data for apoferritin and insulin. This work also highlights that without cooperative experimental and simulative data, development of simulative procedures using MD alone would prove most challenging. Understanding the stability and activity of freeze-dried bio-macromolecules at low degrees of hydration is crucial for pharmaceutical and food industries, however, the building of in silico models for dynamical studies at a molecular level needs careful consideration. Here, the authors propose a modelling protocol that mimics experimental protein lyophilization, and proteins in weakly hydrated amorphous states, and validate it against experimental neutron scattering data.