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

Abstract

a b s t r a c tThe 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.

Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

“…Time resolved fluorescence was measured at room temperature with a streak camera setup as described in detail in ref 16. In brief, the sample is excited by ∼0.2 ps duration pulses (340 nm, ∼1 mW) at a repetition rate of 250 kHz.…”

Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy

“…A single cysteine replaces individual amino acids along the N-terminal region, which is labeled with either a fluorescence (van Oort et al 2009) or a spin-label probe (Kavalenka et al 2009). …”

“…Labeling CP29 at amino acid positions 4, 40 and 97 with a fluorescent TAMRA probe allowed the measurement of ultrafast excitation energy transfer (FRET) from the probe to the Chl (van Oort et al 2009). It turned out that transfer from the probe attached at position 4 was as fast (~20 ps) as from the probe attached at position 97, which is located close to the transmembrane pigment-containing part of the protein, demonstrating that the last stretch of the N-terminus is folded back on the hydrophobic core.…”

Studying fast dynamics in biological complexes : from photosynthesis in vivo to single DNA molecules in vitro

“…Taken together with experimental results using ultrafast spectroscopic techniques, extensive knowledge about the organization and composition of these pigments in thylakoid membranes has been gained highlighting where light absorption, energy transfer and charge separation takes place. Ultrafast spectroscopy is an important experimental technique to characterize these highly efficient processes and identify the important constituents of photosynthetic machinery , van Oort et al 2009, Wientjes et al 2013. In Chapter 4 and 5, we investigated in great detail the ultrafast processes in photosynthetic complexes such as excitation energy transfer (EET) and non-photochemical quenching (NPQ) by using picosecond time-resolved spectroscopy.…”

“…To perform time-resolved picosecond fluorescence measurements, a streak camera system was used as previously described in (van Oort et al 2008a, van Oort et al 2009, van Stokkum et al 2006. The advantage of using a streak camera instead of a more common time-correlated single photon counting setup is that it provides entire spectra with a high temporal resolution.…”