GFP-mut2 proteins in trehalose-water matrixes: spatially heterogeneous protein-water-sugar structures

We report investigations on the properties of nanoenvironments around single-GFP-mut2 proteins in trehalosewater matrixes. Single-GFPmut2 molecules embedded in thin trehalose-water. lms were characterized in terms of their. uorescence brightness, bleaching dynamics, excited state lifetime, and. uorescence polarization. For each property, sets of similar to 100-150 single molecules have been investigated as a function of trehalose content and hydration. Three distinct and interconverting families of proteins have been found which differ widely in terms of bleaching dynamics, brightness, and. uorescence polarization, whose relative populations sizably depend on sample hydration. The reported results evidence the simultaneous presence of different protein-trehalose-water nanostructures whose rigidity increases by lowering the sample hydration. Such spatial inhomogeneity is in line with the well-known heterogeneous dynamics in supercooled. uids and in nonsolid carbohydrate glasses and gives a pictorial representation of the sharp, sudden reorganization of the above structures after uptake reversible arrow release of water molecules.