A Reversibly Photodissociable Split GFP

Abstract

Photocaged molecules, including split Green Fluorescent Protein (GFP), allow for light-activation of in vivo processes or cargo delivery in a minimally invasive manner while offering precise spatial and temporal resolution. GFP has been circularly permuted to have an N-terminal β-strand (strand 7, s7) with a protease cleavage site inserted in a loop added between s7 and the rest of the protein. After cleavage of the loop to create the split protein, the s7 peptide does not dissociate measurably from GFP. In the presence of light, however, s7 photodissociates from the rest of the protein, resulting in a change in UV-Vis absorbance and diminished fluorescence, similar to a previously reported photodissociable split GFP involving β-strand 10 [1]. The rate of photodissociation depends linearly on the number of incident photons until a light-saturated regime is reached, indicating that photodissociation is a single-photon process. In addition, the rate is a function of temperature, indicating that the rate-limiting step is a thermal dissociation step. Re-association with the missing strand occurs spontaneously without the addition of light to form GFP with spectral properties indistinguishable from the original protein. While split GFP has previously been designed for use as a genetically encoded fluorescence reporter of protein-protein interactions [2], this new s7 split GFP represents the next generation of photocaged molecules, creating the opportunity for active control of protein-protein interactions with light.1. Do, K.; Boxer, S. G., J. Am. Chem. Soc., 2011,133, 18078–18081.2. Cabantous et al., Scientific Reports, 2013, 3, 2854.