Environment effects on the oscillatory unfolding kinetics of GFP.
The chromophore of a green fluorescent protein (GFP) mutant engineered to enhance emission and stability is known to display erratic switchings among a few of its chemical substates and, in particular, between the anionic A and the neutral N substates, whose difference is associated with a proton exchange and a consequent conformation rearrangement. However, when close to unfolding, the A-N switchings suddenly become very regular as shown by fluorescence oscillations that have been recently observed for molecules embedded in wet silica gel. In order to establish whether the matrix hosting the protein is responsible for these oscillations, we investigated the effect of another medium (silanized surfaces), of a different denaturant (urea) and of cosolvents (D(2)O and glycerol). The occurrence of periodic A-N switchings, in the last milliseconds before GFP unfolding, is observed under all investigated conditions, together with three specific frequency values that characterize the pre-unfolding fluorescence. Urea and guanidinium, the denaturants employed in order to unfold GFP, do not lead to appreciable differences in the observed switching parameters, whereas the different media embedding the protein give rise only to frequency shifts that scale with the viscosity of the host. The periodicity of the GFP A-N switchings and their dependence on cosolvents suggest that they could be associated with oscillatory motions between meta-stable conformations of the beta-barrel surrounding the chromophore near protein unfolding.
Cannone
Fabio
F
Department of Physics, University of Milano-Bicocca, Milan, Italy.
Collini
Maddalena
M
Chirico
Giuseppe
G
Baldini
Giancarlo
G
Bettati
Stefano
S
Campanini
Barbara
B
Mozzarelli
Andrea
A
D017666:Deuterium Oxide / Q000737:chemistry; D005453:Fluorescence; D005898:Glass / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D017510:Protein Folding; D012821:Silanes / Q000737:chemistry; D013499:Surface Properties; D014508:Urea / Q000737:chemistry
D012821:Silanes; D049452:Green Fluorescent Proteins; D014508:Urea; D017666:Deuterium Oxide; D005990:Glycerol