Ultrafast resonance energy transfer from a site-specifically attached fluorescent chromophore reveals the folding of the N-terminal domain of CP29

The photosynthetic minor antenna complex CP29 of higher plants was singly mutated, overexpressed in Escherichia coli, selectively labeled with the fluorescent dye TAMRA at three positions in the N-terminal domain, and reconstituted with its natural pigments. Picosecond fluorescence experiments revealed rapid excitation energy transfer (∼20 ps) from TAMRA covalently attached to a cysteine at either position 4 or 97 (near the beginning and end of the N-terminal domain) to the chlorophylls in the hydrophobic part of the protein. This indicates that the N-terminus is folded back on the hydrophobic core. In 20% of the complexes, efficient transfer was lacking, indicating that the N-terminus can adopt different conformations. Time-resolved polarized fluorescence measurements demonstrate that the non-transferring conformations only allow restricted rotational motion of the dye molecule. When TAMRA was attached to a cysteine at position 40, the overall transfer efficiency was far lower, reflecting a larger distance to the hydrophobic region.