MbCo in Saccharide Solid Amorphous Systems: A Combined FTIR and SAXS Study

Abstract

Saccharides, and in particular trehalose, are known for their efficiency in protecting biostructures against environmental stress [1], although the preservation mechanism is still debated. Experiments and simulations [2,3] on carboxy-myoglobin (MbCO) showed that the protein dynamics is highly inhibited in dry trehalose matrices, the inhibition being dependent on the water content. In these conditions, a mutual protein-matrix structural and dynamic influence is observed.Here we report a combined FTIR and SAXS study on MbCO embedded in dry amorphous matrices of trehalose and sucrose. FTIR measurements were performed at different protein/sugar ratios, focussing on the stretching band of the bound CO (COB) and on the Water Association Band (WAB), which are spectroscopic markers of the protein and the matrix, respectively.Although the two sugar matrices play similar effects on the COB, large differences are evident in the WAB, which we ascribe to perturbations in the hydrogen-bond network strength. SAXS data confirm these differences: water-dependent domains occur in protein-trehalose systems, which are absent in protein-sucrose systems and in the absence of protein. The comparison between SAXS and FTIR data allow to assign this feature to protein-poor regions, which could better incorporate water than the protein-rich background. These domains could play a buffering action against moisture variations. The reported findings may help in rationalizing the superior trehalose protective effects in terms of structural properties of the whole protein-sugar system.[1] J. H. Crowe, Adv. Exp. Med. Biol. 594, 143-158 (2007).[2] L. Cordone et al., BBA-Prot. Proteom. 1749, 252-281 (2005).[3] S. Giuffrida, G. Cottone, L. Cordone, Biophys. J. 91, 968-980 (2006).