We developed split GFPs as a versatile system for introducing unnatural amino acids.  We found that the best way to do this was to introduce a loop containing a unique proteolytic cleavage site prior to the secondary structure element that would be removed, express the protein with the mature chromophore, remove the part to be replaced by cleavage followed by denaturation, and then refold in the presence of the added synthetic element. By circular permutation, any beta strand or the central helix can be altered.  While studying the refolding process with strand 11, we discovered that reassembly is prevented by chromophore isomerization, but that this can be revived by light as the result of trans->cis photoisomerization. Parallel studies with strand 10 (s10) revealed quite different behavior: strand 10 can be 'photodissociated', i.e. this system is a genetically encoded caged protein.  Exploiting this concept, a novel set of GFP variants that carry one extra s10 were created and characterized. These proteins can fold with either one or the other s10 and can be distinguished by their color.  Possible applications of all of each system as optogenetic tools will be described.

GFP does ESPT - split GFPs do many things

248th National Meeting (2014)

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