We have studied the unfolding of single molecules of GFP-mut2 mutant trapped in wet silica gels in a wide range of GuHCl concentration. After the addition of denaturant, the number of fluorescent molecules decreases with unfolding rates (of the order of 0.01 min(-1)) that are in very good agreement with bulk fluorescence and circular dichroism data. Unexpectedly, single molecule experiments show rare fluctuations in the number of fluorescent proteins at equilibrium. On the other hand, although a first approximate description of the number decays can be reasonably performed by single exponential functions, the distributions of the single molecule unfolding times show a maximum at times congruent to 50-100 min up to the denaturation midpoint concentration of [GuHCl] congruent to 2.5 M. A theoretical analysis of the distributions indicates that this feature is a fingerprint of the competition between unfolding and refolding processes when the protein is very far from the midpoint denaturant concentration.

Chirico, G., Cannone, F., Diaspro, A. (2006). Unfolding time distribution of GFP by single molecule fluorescence spectroscopy. EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, 35(8), 663-674 [10.1007/s00249-006-0075-5].

Unfolding time distribution of GFP by single molecule fluorescence spectroscopy

CHIRICO, GIUSEPPE;
2006

Abstract

We have studied the unfolding of single molecules of GFP-mut2 mutant trapped in wet silica gels in a wide range of GuHCl concentration. After the addition of denaturant, the number of fluorescent molecules decreases with unfolding rates (of the order of 0.01 min(-1)) that are in very good agreement with bulk fluorescence and circular dichroism data. Unexpectedly, single molecule experiments show rare fluctuations in the number of fluorescent proteins at equilibrium. On the other hand, although a first approximate description of the number decays can be reasonably performed by single exponential functions, the distributions of the single molecule unfolding times show a maximum at times congruent to 50-100 min up to the denaturation midpoint concentration of [GuHCl] congruent to 2.5 M. A theoretical analysis of the distributions indicates that this feature is a fingerprint of the competition between unfolding and refolding processes when the protein is very far from the midpoint denaturant concentration.
Articolo in rivista - Articolo scientifico
GFP; two photon microscopy; single molecule; protein unfolding
English
ott-2006
35
8
663
674
none
Chirico, G., Cannone, F., Diaspro, A. (2006). Unfolding time distribution of GFP by single molecule fluorescence spectroscopy. EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, 35(8), 663-674 [10.1007/s00249-006-0075-5].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/3009
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