Tracking Unfolding and Refolding of Single GFPmut2 Molecules

¹ Department of Physics, University of Milan Bicocca, 20126 Milan, Italy.
² Department of Biochemistry and Molecular Biology, Parma, Italy
³ MicroScoBio Reserch Center, University of Genoa, Genoa, Italy
⁴ Department of Public Health, University of Parma, Parma, Italy.
⁵ istituto nazionale per la fisica della materia

^* To whom correspondence should be addressed. E-mail: giuseppe.chirico{at}mib.infn.it.

Submitted on April 14, 2005
Revised on June 3, 2005
Accepted on 17 June 2005

	Abstract

The unfolding and refolding kinetics of more than 600 single GFPmut2 molecules, entrapped in wet nanoporous silica gels, were followed by monitoring simultaneously the fluorescence emission of the anionic and neutral state of the chromophore, primed by two-photon excitation. The rate of unfolding, induced by guanidinium chloride, was determined by counting the number of single molecules that disappear in fluorescence images, under conditions that do not cause bleaching nor photo-induced conversion between chromophore protonation states. The unfolding rate is of the order of 0.01 min-1, and its dependence on denaturant concentration is very similar to that previously reported for high protein load gels. Upon rinsing the gels with denaturant-free buffer, the GFPmut2 molecules refold with rates > 10 min-1, with an apparently random distribution between neutral and anionic states, that can be very different from the pre-unfolding equilibrium. A subsequent very slow (lifetime of about 70 min) relaxation leads to the equilibrium distribution of the protonation states. This mechanism, involving one or more native-like refolding intermediates, is likely rate-limited by conformational rearrangements that are undetectable in circular dichroism experiments. Several unfolding/refolding cycles can be followed on the same molecules, indicating full reversibility of the process and, noticeably, a bias of denaturated molecules towards refolding in the original protonation state.

Key Words: green fluorescent protein, protein encapsulation, protein unfolding, silica gels, single molecule, two photon microscopy

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