Photosystem I from Synechococcus elongatus: preparation and crystallization of monomers with varying subunit compositions

Jekow, Petra; Fromme, Petra; Witt, H. T.

doi:10.1016/0005-2728(94)00201-f

Cited by 23 publications

(14 citation statements)

References 19 publications

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“…Trimeric PS I complexes containing ϳ100 Chl/P700 were prepared from the thermophilic cyanobacterium Synechococcus elongatus as described by Witt et al (1987), except that PS I was extracted with 0.6% (w/w) n-dodecyl-␤,D-maltoside (␤-DM) and purified by anion exchange chromatography on a Q-Sepharose High-Performance column (Pharmacia), as described by Jekow et al (1995). Monomeric PS I complexes containing ϳ95 Chl/P700 were obtained by applying an additional osmotic shock before the purification (Jekow et al, 1995). The purified PS I complexes were suspended in a buffer containing 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.4), 0.02% (w/w) ␤-DM, and ϳ40 mM MgSO 4 (in the case of PS I monomers) or 100 mM MgSO 4 (in the case of PS I trimers) and stored at Ϫ30°C.…”

Section: Methodsmentioning

confidence: 99%

Energy Transfer and Charge Separation in Photosystem I: P700 Oxidation Upon Selective Excitation of the Long-Wavelength Antenna Chlorophylls of Synechococcus elongatus

Pålsson

Flemming

Gobets

et al. 1998

Biophysical Journal

173

201

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Photosystem I of the cyanobacterium Synechococcus elongatus contains two spectral pools of chlorophylls called C-708 and C-719 that absorb at longer wavelengths than the primary electron donor P700. We investigated the relative quantum yields of photochemical charge separation and fluorescence as a function of excitation wavelength and temperature in trimeric and monomeric photosystem I complexes of this cyanobacterium. The monomeric complexes are characterized by a reduced content of the C-719 spectral form. At room temperature, an analysis of the wavelength dependence of P700 oxidation indicated that all absorbed light, even of wavelengths of up to 750 nm, has the same probability of resulting in a stable P700 photooxidation. Upon cooling from 295 K to 5 K, the nonselectively excited steady-state emission increased by 11- and 16-fold in the trimeric and monomeric complexes, respectively, whereas the quantum yield of P700 oxidation decreased 2.2- and 1.7-fold. Fluorescence excitation spectra at 5 K indicate that the fluorescence quantum yield further increases upon scanning of the excitation wavelength from 690 nm to 710 nm, whereas the quantum yield of P700 oxidation decreases significantly upon excitation at wavelengths longer than 700 nm. Based on these findings, we conclude that at 5 K the excited state is not equilibrated over the antenna before charge separation occurs, and that approximately 50% of the excitations reach P700 before they become irreversibly trapped on one of the long-wavelength antenna pigments. Possible spatial organizations of the long-wavelength antenna pigments in the three-dimensional structure of photosystem I are discussed.

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Section: Methodsmentioning

confidence: 99%

Energy Transfer and Charge Separation in Photosystem I: P700 Oxidation Upon Selective Excitation of the Long-Wavelength Antenna Chlorophylls of Synechococcus elongatus

Pålsson

Flemming

Gobets

et al. 1998

Biophysical Journal

173

201

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“…were isolated according to the method of Witt et al I151. PS I monomers were obtained from trimers by a new, very mild procedure as described in [16]. Both PS I complexes contain 8&100 ChUP700.…”

Section: Samples and Biochemical Methodsmentioning

confidence: 99%

Spectroscopic characterization of PS I core complexes from thermophilic Synechococcus sp

et al. 1994

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Monomeric and trimeric PS I complexes missing the three stromal subunits E,C and D (termed PS I core complexes) were prepared from the thermophilic cyanobacterium Synechococcus sp. by incubation with urea. The subunits E,C and D are sequentially removed. In the monomeric PS I the subunit C is removed with a half life of approx. 5 min. This is about eight times faster than in the trimeric PS I complex. In parallel with the removal of the FAB containing subunit C the reduction kinetics of P700+ changed from a half life of about 25 ms to about 750 μs. The partner of P700+ in the 750 μs charge recombination was identified to be FX by the difference spectrum of this phase. There are some minor differences in the spectra of trimeric and monomeric PS I core complexes. At 77K the forward electron transfer from A − 1 to FX is blocked in the major fraction of the PS I core complexes and P700+A− 1 recombines with a half life of about 220 μs. In the remaining fraction P700+FX − is formed and decays with a half life of approx. 10 ms at 77 K. The kinetics of the forward electron transfer from A− 1 to the iron‐sulfur‐clusters was measured in the native PS I and the corresponding core complexes. The reoxidation kinetics of A− 1 are identical in both cases (t 12 = 180 ns). We conclude that Fx is an obligatory intermediate in the normal forward electron transfer.

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“…Although other well characterized cyanobacterial PS I preparations exist from Synechocystis PCC 6803 (3,19,24) or S. elongatus (43), for instance, they do not offer these possibilities. Especially with Synechococcus it is extremely difficult to isolate monomers with the complete set of all subunits, particularly the PsaL protein (35). In Synechocystis, which can be easily genetically modified, the trimers seem to be less stable.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Oligomerization of a Membrane Protein Complex

Kruip

Karapetyan

Терехова

et al. 1999

Journal of Biological Chemistry

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Many membrane proteins can be isolated in different oligomeric forms. Photosystem I (PSI), for example, exists in cyanobacteria either as a monomeric or as a trimeric complex. Neither the factors responsible for the specific trimerization process nor its biological role are known at present. In the filamentous cyanobacterium Spirulina platensis, trimers in contrast to monomers show chlorophyll fluorescence emission at 760 nm. To investigate the oligomerization process as well as the nature of the long wavelength chlorophylls, we describe here an in vitro reconstitution procedure to assemble trimeric PS I from isolated purified PS I monomers. Monomers (and trimers) were extracted from S. platensis with n-dodecyl ␤-D-maltoside and further purified by perfusion chromatography steps. The isolated complexes had the same polypeptide composition as other cyanobacteria (PsaA-PsaF and PsaI-PsaM), as determined from high resolution gels and immunoblotting. They were incorporated into proteoliposomes, which had been prepared by the detergent absorption method, starting from a phosphatidylcholine:phosphatidic acid mixture solubilized by octylglucoside. After the addition of monomeric PS I (lipid:chlorophyll, 25:1), octylglucoside was gradually removed by the stepwise addition of Biobeads. The 77 K fluorescence emission spectrum of these proteoliposomes displays a long wavelength emission at 760 nm that is characteristic of PS I trimers, which indicates for the first time the successful in vitro reconstitution of PS I trimers. In addition, a high performance liquid chromatography analysis of complexes extracted from these proteoliposomes confirms the formation of structural trimers. We also could show with this system 1) that at least one of the stromal subunits PsaC, -D, and -E is necessary for trimer formation and 2) that the extreme long wavelength emitting chlorophyll is formed as a result of trimer formation.

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Photosystem I from Synechococcus elongatus: preparation and crystallization of monomers with varying subunit compositions

Cited by 23 publications

References 19 publications

Energy Transfer and Charge Separation in Photosystem I: P700 Oxidation Upon Selective Excitation of the Long-Wavelength Antenna Chlorophylls of Synechococcus elongatus

Energy Transfer and Charge Separation in Photosystem I: P700 Oxidation Upon Selective Excitation of the Long-Wavelength Antenna Chlorophylls of Synechococcus elongatus

Spectroscopic characterization of PS I core complexes from thermophilic Synechococcus sp

In Vitro Oligomerization of a Membrane Protein Complex

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