Proteins undergo a dynamic transition at approximately 220 K, also called protein ’glass’ transition, below which temperature proteins lose their conformational flexibility and become biologically inactive. The protein dynamic transition has been observed for several proteins utilizing different techniques, such as inelastic neutron scattering, infrared spectroscopy and X-ray crystallography. Water seems to play a role in this transition, especially in relation to the hydration layer surrounding the protein, termed hydration water, whose molecules interact with the protein surface. Despite various interpretations that have been advanced, the origin of the protein dynamic transition is still elusive.

Here, we investigate the protein ’glass’ transition by means of infrared spectroscopy and X-ray diffraction, focusing on the enzyme lysozyme over the temperature range 160-295K. Starting with the experimental implementation of the protein powder hydration setup, we study different levels of hydration of the protein, comparing them with the dry lysozyme as well as with protein solutions of different concentrations. We find a crossover in the lysozyme structure that occurs near 230 K, enhanced by the presence of hydration water.