GFP-mut2 Proteins in Trehalose-Water Matrixes: Spatially Heterogeneous Protein-Water-Sugar Structures
Laura D'Alfonso 1, Maddalena Collini 1, Fabio Cannone 1, Giuseppe Chirico 1, Barbara Campanini 2, Grazia Cottone 3 and Lorenzo Cordone 3*
1 Dipartimento di Fisica, Università di Milano Bicocca
2 Dipartimento di Biochimica e Biologia Molecolare, Università di Parma
3 Dipartimento di Scienze Fisiche ed Astronomiche, Università di Palermo
* To whom correspondence should be addressed. E-mail: cordone{at}fisica.unipa.it.
Submitted on June 6, 2006
Revised on August 25, 2006
Accepted on 5 February 2007
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Abstract |
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We report investigations on the properties of nano-environments 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 about 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 are widely differing 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 non-solid carbohydrate glasses, and gives a pictorial representation of the sharp, sudden reorganisation of the above structures following uptake 
release of water molecules.
Key Words:
Green Fluorescent Protein, heterogeneous dynamics, single molecule, trehalose, two photons excitation (TPE)
