Excerpts

The optimized photoconvertible fluorophores C3PA-GFP and SPA-GFP were identified as part of a screen of mutations in the canonical photoconvertible fluorophore PA-GFP with improved diffusional and folding properties. The screen (details of which are available from S.R.D.) included the analysis of previously described mutants of GFP for enhanced photoconversion in our system. C3PA-GFP incorporates three additional mutations (F99S, M153T and V163A) initially identified in the ‘Cycle 3’ mutant of GFP. V163A was originally incorporated into the first-generation PA-GFP molecule. SPA-GFP incorporates nine mutations (S30R, Y39N, A206V, M153T, Y145F, V163A, I171V, F99S and N105T) identified in the ‘Superfolder’ mutant of eGFP. The mutations in C3PA-GFP and SPA-GFP do not include the ‘eGFP’ mutations F64L and S65T, which disrupt the ability of PA-GFP to undergo photoconversion.