Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy.
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 (approximately 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 (approximately 15 A), 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.
Berghuis
Bojk A
BA
Laboratory of Biophysics, Wageningen University, 6700 ET Wageningen, The Netherlands.
Spruijt
Ruud B
RB
Koehorst
Rob B M
RB
van Hoek
Arie
A
Laptenok
Sergey P
SP
van Oort
Bart
B
van Amerongen
Herbert
H
D019943:Amino Acid Substitution; D016477:Artifacts; D001896:Boron Compounds / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry* / Q000235:genetics; D017785:Photons; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000235:genetics; D011336:Probability; D017434:Protein Structure, Tertiary; D013997:Time Factors
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; C069660:CP29 light harvesting complex; D005456:Fluorescent Dyes; D045342:Light-Harvesting Protein Complexes; D045332:Photosystem II Protein Complex