β-Relaxation governs protein stability in sugar-glass matrices

The stabilizing effect of sugar-glass matrix materials for freeze-drying proteins or nucleic acids has been variously ascribed to the thermodynamic effect of ‘water replacement’ by sugar molecules or to the kinetic effect of slowed α relaxation associated with sugar matrix vitrification. While evidence for each of these hypotheses exists, we show that neither can adequately account for the observed stabilization of proteins embedded in sugar-glasses. Instead, we find firm evidence that protein stability in these glasses is directly linked to high frequency β relaxation processes of the sugar matrix. Specifically, we observe that when the β relaxation time, τβ, of sugar-glasses is increased with antiplasticizing additives, protein stability increases in linear proportion to the increase in τβ, even though these same additives simultaneously decrease the glass transition temperature, Tg, and the α relaxation time, τα, of the sugar matrix materials. Moreover, we find that while sugars ‘replace’ water by stabilizing protein native-like conformation in the dry state, the resulting enhanced protein conformational stability does not have a significant impact on the degradation rate of the proteins in sugar-glasses. We discuss implications of these findings for the fundamental physics of glass formation and for effective engineering of protein stabilizing glasses through the modification of τβ.