Abstract
The light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCIIb) fulfills multiple functions, such as light harvesting and energy dissipation under different illuminations. The crystal structure of LHCIIb at the near atomic resolution reveals an antiparallel strands structure in the lumenal loop between the transmembrane helices B/C. To study the structural and functional significances of this structure, three amino acids (Val-119, His-120, and Ser-123) in this region have been exchanged to Phe, Leu, and Gly, respectively, and the influence of the mutagenesis on the structure and function of LHCIIb has been investigated. The results are as follows. 1) Circular dichroism spectra of the mutations reveals that the antiparallel strands in the lumenal region are very important for adjusting pigment conformation in the neoxanthin domain of LHCIIb. Although the mutagenesis causes only a slight loss of the Neo binding in the complexes (V119F, 0.09; S123G, 0.19; and H120L, 0.27), it imparts remarkable changes to the pigment conformation. 2) Substituting Ser-123 with Gly results in a higher susceptibility to photodamage, an increased tendency to aggregate, and enhanced fluorescence quenching induced by the medium acidification. These results demonstrate that this antiparallel strands domain plays an important role in regulating the pigment conformation and in adjusting the aggregation and the fluorescence yield of LHCIIb.
Highlights
As is revealed by the LHCIIb crystal structure at near atomic resolution [5, 14], LHCIIb is composed of three homo- or hetero monomers, each binding 14 Chls (8 Chl a, 6 Chl b), 4 carotenoid molecules (2 luteins (Lut), one neoxanthin (Neo), one violaxanthin (Vio) (or zeaxanthin (Zea)), and 2 lipids
LHCIIb provides a unique opportunity for studies of the roles of individual amino acids or particular structural features of the protein because LHCIIb is one of only a handful of membrane proteins that can be refolded in vitro [23, 40, 41], and still maintain characteristics that are similar to the native ones in vivo as demonstrated by detailed spectroscopic and biochemical analyses [33, 42], and for which a high-resolution structure is known [5, 14]
Our results demonstrate that the characteristics of these amino acid residues (Val-119, His-120, and Ser-123) are not indispensable for refolding and maintenance of the thermal stability of the pigment protein complexes, because the mutations can bind pigments in the in vitro reconstitution system and refold correctly into functional LHCIIb complexes with similar thermal stability (Table 3) and ␣-helix content in the complex (Supplemental Table 1) to the wild type (WT) LHCIIb
Summary
As is revealed by the LHCIIb crystal structure at near atomic resolution [5, 14], LHCIIb is composed of three homo- or hetero monomers, each binding 14 Chls (8 Chl a, 6 Chl b), 4 carotenoid molecules (2 luteins (Lut), one neoxanthin (Neo), one violaxanthin (Vio) (or zeaxanthin (Zea)), and 2 lipids. Neo has the lowest efficiency of energy transfer to Chl in all carotenoids [17], but it is a good scavenger for singlet O2*, which is very important for photoprotection in PSII [18] It has been observed with recombinant LHCIIb that Neo is subject to reversible dissociation under different high ambient temperatures despite its high affinity to LHCIIb [19]. The isomerism of all-trans Vio to 9-cis Vio, which replaces 9-cis Neo in the N1-position, could be induced under elevated temperatures in native LHCIIb, isolated from winter rye leaves [21]. It suggests that a 9-cis-5,6-epoxy-3-hydroxy carotenoid is required for this position. The changes in pigment stoichiometries, characteristics of optical spectra, capacity for non-radiative dissipation, the characteristics of macro structure in a low detergent medium, and the characteristics of low pH-induced fluorescence lowering of different LHCIIb species have been investigated, and the possible functional mechanisms of the motif are discussed in this article
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