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GFP-mut2 Proteins in Trehalose-Water Matrixes: Spatially Heterogeneous Protein-Water-Sugar Structures

Laura D'Alfonso ^* ¶, Maddalena Collini ^* ¶, Fabio Cannone ^* ¶, Giuseppe Chirico ^* ¶, Barbara Campanini  , Grazia Cottone  ^¶ and Lorenzo Cordone  ^¶

^* Dipartimento di Fisica, Università di Milano Bicocca, Milano, Italy; Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Parma, Italy; Dipartimento di Sanità Pubblica, Università di Parma, Parma, Italy; Dipartimento di Scienze Fisiche e Astronomiche, Università di Palermo, Palermo, Italy; and ^¶ Consorzio Nazionale Interuniversitario Per Le Scienze Fisiche Della Materia, Roma, Italy

Correspondence: Address reprint requests to Lorenzo Cordone, E-mail: cordone{at}fisica.unipa.it.

We report investigations on the properties of nanoenvironments around single-GFP-mut2 proteins in trehalose-water matrixes. Single-GFPmut2 molecules embedded in thin trehalose-water films were characterized in terms of their fluorescence brightness, bleaching dynamics, excited state lifetime, and fluorescence polarization. For each property, sets of 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 fluorescence 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 fluids and in nonsolid carbohydrate glasses and gives a pictorial representation of the sharp, sudden reorganization of the above structures after uptake release of water molecules.