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

Abstract

A high-throughput Förster resonance energy transfer (FRET) study was performed on the approximately 100 amino acids long N-terminal domain of the photosynthetic complex CP29 of higher plants. For this purpose, CP29 was singly mutated along its N-terminal domain, replacing one-by-one native amino acids by a cysteine, which was labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP9, chlorophylls and xanthophylls. Picosecond fluorescence experiments revealed rapid energy transfer (~20–70 ps) from BODIPY at amino-acid positions 4, 22, 33, 40, 56, 65, 74, 90, and 97 to Chl a molecules in the hydrophobic part of the protein. From the energy transfer times, distances were estimated between label and chlorophyll molecules, using the Förster equation. When the label was attached to amino acids 4, 56, and 97, it was found to be located very close to the protein core (~15 Å), whereas labels at positions 15, 22, 33, 40, 65, 74, and 90 were found at somewhat larger distances. It is concluded that the entire N-terminal domain is in close contact with the hydrophobic core and that there is no loop sticking out into the stroma. Most of the results support a recently proposed topological model for the N-terminus of CP29, which was based on electron-spin-resonance measurements on spin-labeled CP29 with and without its natural pigment content. The present results lead to a slight refinement of that model.

Exploring the Structure of the 100 Amino-Acid Residue Long N-Terminus of the Plant Antenna Protein CP29

“…The construction and isolation of overexpressed CP29 apoprotein from Lhcb4.1 cDNA of Arabidopsis thaliana (from the Arabidopsis Biological Resource Center DNA Stock Center) were performed as reported previously (8,9). The naturally occurring cysteine (position 108) was replaced by alanine.…”

“…The naturally occurring cysteine (position 108) was replaced by alanine. The mutant protein (C108A) was used to estimate the amount of nonspecific spin labeling (8,9). Single cysteine mutants were introduced at 55 different positions in the N-terminus of the CP29 apoprotein using this template, resulting in the following mutants: G4C, G6C, A10C, A11C, S15C , A16C, T19C, V20C, T21C, T22C, P29C, G30C, A31C, I32C, S33C, G39C, S40C, L41C, V42C, G43C, G46C, F50C, G51C, L52C, G53C, A56C, E57C, Y58C, L59C, Q60C, F61C, S65C, Q68C, N69C, L70C, A71C, N73C, L74C, A75C, G76C, G80C, T81C, T83C, E84C, A85C, A86C, A88C, S90C, T91C, P92C, F93C, Q94C, S97C, G101C, and the wild-type protein, labeled for consistency as C108C.…”

“…In the DEER experiments, we investigated three different double cysteine mutants: A56C/S65C, A56C/T81C, and A56C/S97C. Pigment isolation, labeling, and reconstitution of CP29 pigment complexes were performed as described previously (8,9). Solutions of the spin-labeled CP29 samples were washed and concentrated in sucrose-free DM buffer (0.03% W/V þ 10 mM Na 2 HPO 4 , pH 7.6) just before the EPR measurements were obtained.…”

“…In PSII, the outer antenna system is built from a large number of complexes, the most relevant of which for this work are LHCII, the major antenna subunit, and three minor complexes, CP24, CP26, and CP29 (1,3). Among the minor complexes, CP29 is the largest one and is believed to collect, conduct, and possibly quench electronic excitation energy in photosynthesis (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Moreover, CP29 is essential for the proper assembly of PSII and its absence leads to a large drop in the average rate of excitation trapping in the reaction centers of PSII (10).…”

“…Therefore the better-characterized LHCII cannot be used as a model for the entire N-terminus of CP29. Previous studies that targeted the structure of the N-terminus of CP29 by fluorescence resonance energy transfer (FRET) (8,10) and EPR (9) indicated that the N-terminus is folded back onto the protein, and suggested the presence of some structural heterogeneity in the N-terminal part.…”