Journal of Physical Chemistry Letters
Journal of Physical Chemistry Letters
5 years ago

A Double Decarboxylation in Superfolder Green Fluorescent Protein Leads to High Contrast Photoactivation

A Double Decarboxylation in Superfolder Green Fluorescent Protein Leads to High Contrast Photoactivation
Joshua D. Slocum, Lauren J. Webb
A photoactivatable variant of superfolder green fluorescent protein (GFP) was created by replacing the threonine at position 203 with aspartic acid. Photoactivation by exposure of this mutant to UV light resulted in conversion of the fluorophore from the neutral to the negatively charged form, accompanied by a ∼95-fold increase in fluorescence under 488 nm excitation. Mass spectrometry before and after exposure to UV light revealed a change in mass of 88 Da, attributed to the double decarboxylation of Glu 222 and Asp 203. Kinetics studies and nonlinear power-dependence of the initial rate of photoconversion indicated that the double decarboxylation occurred via a multiphoton absorption process at 254 nm. In addition to providing a photoactivatable GFP with robust folding properties, a detailed mechanistic understanding of this double decarboxylation in GFP will lead to a better understanding of charge transfer in fluorescent proteins.

Publisher URL: http://dx.doi.org/10.1021/acs.jpclett.7b01101

DOI: 10.1021/acs.jpclett.7b01101

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