Dynamics of green fluorescent protein mutant2 in solution, on spin-coated glasses, and encapsulated in wet silica gels.
Single-molecule experiments are performed by investigating spectroscopic properties of molecules either diffusing in and out of the observation volume or fixed in space by different immobilization procedures. To evaluate the effect of immobilization methods on the structural and dynamic properties of proteins, a highly fluorescent mutant of the green fluorescent protein, GFPmut2, was spectroscopically characterized in bulk solutions, dispersed on etched glasses, and encapsulated in wet, nanoporous silica gels. The emission spectrum, the fluorescence lifetimes, the anisotropy, and the rotational correlation time of GFPmut2, encapsulated in silica gels, are very similar to those obtained in solution. This finding indicates that the gel matrix does not alter the protein conformation and dynamics. In contrast, the fluorescence lifetimes of GFPmut2 on glasses are two-to fourfold higher and the fluorescence anisotropy decays yield almost no phase shifts. This indicates that the interaction of the protein with the bare glass surface induces a significant structural perturbation and severely restricts the rotational motion. Single molecules of GFPmut2 on glasses or in silica gels, identified by confocal image analysis, show a significant stability to illumination with bleaching times of the order of 90 and 60 sec, respectively. Overall, these data indicate that silica gels represent an ideal matrix for following biologically relevant events at a single molecule level.
Chirico
Giuseppe
G
Department of Physics, University of Milan-Bicocca, 20126 Milan, Italy. giberto.chirico@mib.infn.it
Cannone
Fabio
F
Beretta
Sabrina
S
Diaspro
Alberto
A
Campanini
Barbara
B
Bettati
Stefano
S
Ruotolo
Roberta
R
Mozzarelli
Andrea
A
D016880:Anisotropy; D005898:Glass; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D018613:Microscopy, Confocal; D009154:Mutation; D058428:Silica Gel; D012822:Silicon Dioxide / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D058428:Silica Gel; D012822:Silicon Dioxide