A water-soluble chlorophyll-binding protein (WSCP) is the single known instance of a putative chlorophyll (Chl) carrier in green plants. Recently the photoprotective function of WSCP has been demonstrated by EPR measurements; the light-induced singlet-oxygen formation of Chl in the WSCP tetramer is about four times lower than that of unbound Chl. This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum (Virginia pepperweed) to clarify the mechanism of its photoprotective function. The WSCP-Chl complex is a homotetramer comprising four protein chains of 180 amino acids and four Chl molecules. At the center of the complex one hydrophobic cavity is formed in which all of the four Chl molecules are tightly packed and isolated from bulk solvent. With reference to the novel Chl-binding mode, we propose that the photoprotection mechanism may be based on the inhibition of physical contact between the Chl molecules and molecular oxygen.Water-soluble chlorophyll-binding proteins (WSCPs), 4 which form a complex with chlorophyll (Chl), have been isolated from Amaranthaceae, Chenopodiaceae, and Polygonaceae (class-I) and from Brassicaceae (class-II). The two WSCP classes differ in that the protein-Chl complexes of the former change their absorption spectra upon illumination, whereas those of the latter show no photoconversion of their absorption behavior (2). The amino acid sequence of class-I WSCP shows no similarity to that of class-II WSCP.The class-II WSCPs (hereafter referred to as "WSCPs") are water-soluble proteins of ϳ20 kDa, which form a tetrameric assembly upon binding Chl molecules with a Chl-to-protein ratio of one or less, and exhibit high thermal and photostability (1, 3). The physiological function of WSCPs is still not known. Although WSCPs have a sequence similarity of ϳ30% with Künitz-type proteinase inhibitors, no significant protease inhibitor activity of WSCPs has yet been identified (4 -6). The low Chl content per protein makes it unlikely that WSCPs are involved in the light reaction of photosynthesis. Meanwhile, it has been speculated (7, 8) that WSCP acts as a scavenger of free Chl, by transporting it from the thylakoid membrane to the chloroplast envelope, where Chlase, the enzyme that initiates the Chl catabolism, is thought to reside (9).An in vitro binding assay with the recombinant apo-WSCP cloned from cauliflower (Brassica oleralcea var. Botrys) and Chl or its derivatives showed that the central Mg 2ϩ ion and the phytyl tail of Chl were essential for protein-pigment binding and tetrameric assembly, respectively (1). Furthermore, the recombinant apo-WSCP was able to remove Chl from the thylakoid membrane in vitro and organize the tetrameric WSCPChl complex (3). The absorption spectrum of the reconstituted complex is very similar to that of the native WSCP-Chl complex purified from fresh leaves (1, 3).The WSCP-Chl complex retains its fresh green color under dim light for months, enough for a long term crystallization experiment. Free Chl, on t...

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The medicinal plant Holboellia fargesii REAUB. (Lardizabalaceae) is a tree which grows in the southwest region of the People's Republic of China. Its stems, roots, and fruits have been used as folk medicine for the treatment of cough, lumbago, nephritis and abdominal hernia in Yunnan Province. 1,2) However, there has been no previous chemical study of this plant. Preliminary evaluation indicated that H. fargesii contains saponins. The potential medicinal importance and our continuing interest in the chemistry of saponins prompted us to initiate a chemical investigation of this plant. In this paper, we report the isolation and structure elucidation of five new triterpenoid saponins (1-5) from the roots of this species. Results and DiscussionAn ethanolic extract of roots of H. fargesii was suspended in water and then partitioned successively with EtOAc and nBuOH. The n-BuOH soluble fraction, on chromatographic purification over Diaion HP-20 resin, followed by repeated medium-pressure liquid chromatography (MPLC) and HPLC purification, gave five new triterpenoid saponins, termed fargosides A-E (1-5).Fargoside A (1) gave quasi-molecular ions at m/z 775 [MϩNa] ϩ and 791 [MϩK] ϩ by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS. The molecular formula of C 40 H 64 O 13 was confirmed by high resolution (HR)-FAB-MS. The IR spectrum of 1 showed an absorption bond for a carbonyl group at 1685 cm Ϫ1. The 13 C-NMR spectrum showed 40 carbon signals, of which 29 were assigned to the aglycon moiety and 11 were assigned to the sugar moiety (Tables 1-3 5, 16.5, 17.4, 25.7, 26.1 and 27.9, and sp 2 hybrid carbons at d 122.7 (d) and 144.4 (s), together with information from the 1 H-NMR spectrum (six methyl proton singlets and a broad triplet olefinic proton at d 5.54), indicated that the aglycon had an olean-12-ene skeleton.3) Detailed analysis of various two-dimensional (2D) NMR spectra such as double-quantum filtered 1 H-1 H shift correlation spectroscopy (DQF-COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC) and phase-sensitive nuclear Overhauser enhancement spectroscopy (NOESY) spectra indicated that the structural features in the A, B, C and D rings of the aglycon were similar to those of oleanolic acid. 4) However, direct bond and long-range correlation data suggested that one of the geminal methyl groups at C-20 had been replaced by a hydroxyl group, forming 29-noroleanolic acid. In the NOESY spectrum (Fig. 1), H-18 was correlated with the methyl group at C-20, indicating that the hydroxyl group was in an a-configuration. These results suggested that the aglycon of 1 was 3b,20a-dihydroxy-29-norolean-12-en-28-oic acid. A related compound, 3b,20x-dihydroxy-30(or 29)-