In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET.

Fluorescence resonance energy transfer (FRET) analysis in biological systems has reached broad application along with the fast improvement of fluorescent proteins. This work shows the advancement of the commonly used single-step FRET between two fluorophores to a two-step FRET analysis with three fluorophores in vivo. In addition to CFP and YFP the DsRed derivative mCherry was genetically fused in frame to the coding region of the plastidic 2-Cys peroxiredoxin and co-expressed in plant cells resulting in detectable radiationless energy transfer from CFP via YFP to mCherry. The use of control constructs such as fused fluorophore pairs of CFP, YFP and mCherry, but also YFP:mCherry:CFP and REACh:mCherry:CFP allowed for the generation of a reference matrix for two-step FRET calculations. The occurrence of two-step FRET proves that the obligate 2-Cys peroxiredoxin dimers assemble to higher mass oligomers presumably decamers in vivo. This finding together with previous reports on structural dynamics and functional switching of 2-Cys peroxiredoxin might indicate a conformation linked redox-signalling function of the 2-Cys Prx. Although three different fusion proteins had to be imported by the chloroplast two-step FRET was significant within the 2-Cys peroxiredoxin complex. In addition to the proof of oligomerisation in vivo, the results demonstrate the large potential of the method for investigating tripartite protein interactions in subcellular compartments and in general cell biology.