THE MAJOR INTRINSIC LIGHT-HARVESTING PROTEIN OF Amphidinium: CHARACTERIZATION AND RELATION TO OTHER LIGHT-HARVESTING PROTEINS

A major light-harvesting complex (LHC) has been obtained from thylakoids of Amphidinium carterae solubilized with digitonin or decylmaltoside and separated by sucrose-gradient centrifugation. The digitonin-LHC forms a dark brown band at -17% sucrose and the decylmaltoside LHC one at -7% sucrose. Excellent energy transfer is retained from chlorophyll c and carotenoid to chlorophyll a. Absorbance and fluorescence excitation spectra show the existence of two major forms of chlorophyll c, one absorbing at 634 nm and the other at 649 nm. Linear dichroism spectra show the Q_y transition of both forms of chlorophyll c to be aligned at <35° to the membrane plane. On sodium dodecylsulfate polyacrylamide gels the complex resolves as a single band of 19 kDa. A partial amino acid sequence shows the N-terminus to be unblocked but modified; there is a persistent ambiguity of Ser/Asn at residue 4 and evidence for multiple but very similar polypeptides within the 19 kDa band. The peptide has strong identity with the N-terminal regions of LHC from Phaeodactylum and Pavlova and LHC 1 of higher plants. Antibodies to the 19 kDa peptide react weakly with LHC of brown algae, diatoms and Prymnesiophytes but not with those of higher plants or Cryptophytes.     

CONFIGURATIONS OF CAROTENOIDS IN THE REACTION CENTER and THE LIGHT-HARVESTING COMPLEX OF Rhodospirillum rubrum. NATURAL SELECTION OF CAROTENOID CONFIGURATIONS BY PIGMENT PROTEIN COMPLEXES

Abstract— Carotenoids extracted from the reaction center (RC), the light-harvesting complex (LH), and the chromatophore membrane of Rhodospirillum rubrum SI were analyzed by high-performance liquid chromatography. The chemical structures and the configurations of major components were determined by means of mass, Raman, electronic absorption and ¹H-NMR spectroscopy. The results indicated: (1) 15- cis -spirilloxanthin is bound to RC; (2) both all-frans-spirilloxanthin and aII-(ran.s-3,4-dihydrospirilloxanthin are bound to LH and (3) 13-cK-spirilloxanthin is additionally present in the chromatophore membrane. The natural selection of the carotenoid configurations, i.e. 15-ris by RC and aW-trans by LH, is discussed in relation to the physiological functions and the photophysical properties of isomeric carotenoids.     

NONLINEAR LASERSPECTROSCOPIC INVESTIGATIONS OF THE PIGMENT-PIGMENT INTERACTION WITHIN THE LIGHT-HARVESTING COMPLEX OF PHOTOSYSTEM II

Using a pump and test beam technique in the frequency domain with pump pulses in the nanosecond time range, the nonlinear transmission properties were investigated at room temperature in photosystem (PS) II membrane fragments and isolated light-harvesting chlorophyll a/b-protein preparations (LHC II preparations). In LHC II preparations and PS II membrane fragments, respectively, pump pulses of 620 nm and 647 nm cause a transmission decrease limited to a wavelength region in the nearest vicinity of the pump pulse wavelength (full width at half maximum ' 0.24 nm). In contrast, at 670 nm neither a transmission decrease nor a narrow band feature were observed. The data obtained for PS II membrane fragments and LHC II preparations at shorter wavelengths (620 nm, 647 nm) were interpreted in terms of excited state absorption of whole pigment-protein clusters within the light-harvesting antenna of photosystem II. The interpretation of the small transmission changes as homogeneously broadened lines led to a transversal relaxation time for chlorophyll in the clusters of about 4 ps.     

PIGMENT ORGANIZATION IN THE LIGHT-HARVESTING CHLOROPHYLL-a/b PROTEIN COMPLEX OF LETTUCE CHLOROPLASTS. EVIDENCE OBTAINED FROM PROTECTION OF THE CHLOROPHYLLS AGAINST PROTON ATTACK and FROM EXCITATION ENERGY TRANSFER

Abstract— The light-harvesting Chl-a/b protein complex (LHC) of Lactuca sativa L. was examined for pigment content, excitation energy transfer and behavior under acidic conditions:
(1) Lettuce LHC contains Chl-a, Chl-b and xanthophylls (lutein, neoxanthin, lactucaxanthin, viola-xanthin) at a molar ratio of 6:4:3; their contribution to the absorbance of the LHC between 390 and 530 nm is estimated to be about 31% (Chl-a), 26% (Chl-h) and 43% (xanthophylls).
(2) Energy transfer from xanthophylls and Chl-fe to Chl-a takes place at 100% transfer efficiency.
(3) LHC exhibits an unusual acid stability: in contrast to complexes of photosystem I or II, LHC-bound chlorophylls are not converted to phaeophytin and LHC apoprotein is not denatured at pH 1.5; also, energy transfer is maintained.
(4) Pronase or trypsin treatment do not affect acid stability and energy transfer.
(5) Treatments that break down acid stability (heat, urea or TritonX–100) also inhibit energy transfer.
The coincidental breakdown of energy transfer and acid stability points at one underlying process, namely, the breakdown of a structure that enables protection of chlorophylls from proton attack and close contiguity of xanthophylls and chlorophylls as required for energy transfer. Dense packing of xanthophylls and chlorophylls within lipophilic crevices of the LHC is suggested.     

ISOLATION and CHARACTERIZATION OF A SUBUNIT FORM OF THE B875 LIGHT-HARVESTING COMPLEX FROM Rhodobacter capsulatus

A structural subunit (called B816) has been isolated from the B875 light-harvesting complex of Rhodobacter capsulatus using a detergent-mediated dissociation of chromatophores. Rb. capsulatus MW442 (B800-850- B875+ car+) chromatophores were extracted with benzene and titrated with octyl glucoside (OG) to shift the near-infrared absorption maximum from 873 to 816 nm. Gel filtration chromatography was then used to separate B816 from reaction centers. B816 could be quantitatively shifted back to a B875-like form (lambda max = 875 nm) by decreasing the OG concentration. A similar B816 species could be isolated in low yield from wildtype (B800-850+ B875+ car+) cells but not from SB203E (B800-850- B875+ car-). In the latter case, the B816 subunit seemed too unstable to be isolated under equivalent conditions. The alpha:beta polypeptide ratio, the CD spectrum, and the ability to reversibly dissociate B816 to free bacteriochlorophyll and alpha- and beta-polypeptides were found to be similar to those of the B820 subunit of Rs. rubrum previously reported by our laboratory.     

PIGMENT COMPLEXES OF LIGHT-HARVESTING CHLOROPHYLL a/b BINDING PROTEIN ARE STABILIZED BY A SEGMENT IN THE CARBOXYTERMINAL HYDROPHILIC DOMAIN OF THE PROTEIN*

In order to identify segments of light-harvesting chlorophyll a/6-binding protein (LHCP) that are important for pigment binding, we have tested various LHCP mutants regarding their ability to form stable pigment-protein complexes in an in vitro reconstitution assay. Deletion of 10 C-terminal amino acids in the LHCP precursor, pLHCP, did not significantly affect pigment binding, whereas deletion of one additional amino acid, a tryptophan, completely abolished the formation of stable pigment-protein complexes. This tryptophan, however, can be exchanged with other amino acids in full-length pLHCP without noticeably altering the stability or spectroscopic properties of pigment complexes made with these mutants. Thus, the tryptophan residue is not likely to be involved in a highly specific interaction stabilizing the complex. A double mutant of LHCP lacking 66 N-terminal and 6 C-terminal amino acids still forms pigmented complexes that are virtually identical to those formed with the full-length protein concerning their pigment composition and spectroscopic properties. We conclude that about 30% of the polypeptide chain in LHCP is not involved in pigment binding.     

RHYTHMIC REGULATION OF THE LIGHT-HARVESTING CHLOROPHYLL alb PROTEIN and THE SMALL SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE mRNA IN RYE SEEDLINGS

In etiolated rye seedlings transferred to light the expression of chlorophyll a/b binding protein mRNA varies when the seedlings are grown in a day/night cycle. The fluctuation pattern follows a circadian rhythm. Exposure of 4-day old etiolated seedlings to continuous white light revealed two maxima within the first 24 h before the 24 h cycle period appeared. These first two maxima are also observable after a pulse of white light or after a pulse of red light. These results indicate a possible involvement of phytochrome in the endogenous regulation of the rhythm.     

LINEAR AND CIRCULAR DICHROISM AND FLUORESCENCE POLARIZATION OF THE B875 LIGHT-HARVESTING BACTERIOCHLOROPHYLL-PROTEIN COMPLEX FROM RHODOPSEUDOMONAS SPHAEROIDES

Abstract— The orientation of the chromophores in the B875 light-harvesting bacteriochlorophyll complex isolated from Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis was examined by linear and circular dichroism and fluorescence polarization procedures. The circular dichroism in the near-IR was weaker than that of the B800–850 light-harvesting complex and had a distinctly different shape. This suggested a different geometry for the two bacteriochlorophylls of B875 and less interactive association between their transition moments. The magnitude of the circular dichroism in the carotenoid region of B875 was similar to that of B800–850 but gave more negative values between approx. 430–485 nm; this may reflect a difference in the asymmetric binding of carotenoids to the B875 protein. The fluorescence polarization increased sharply across the near-IR region of B875 and achieved very high values at long wavelengths. This confirmed that more than one transition contributed to this absorption band. The linear dichroism of B875 did not show a significant change in this near-IR band like that observed for the longest wavelength band of B800–850. Thus, the transition moments for each bacteriochlorophyll within B875 appear to be tilted to approximately the same extent with respect to the protein axis. These results distinguish B875 from all other light-harvesting complexes and suggest that the antennae of Rhodospirillaceae which contain a single near-IR absorption band cannot be classified into a single group.     

ALTERED SPECTRAL PROPERTIES OF THE B875 LIGHT-HARVESTING PIGMENT-PROTEIN COMPLEX IN A Rhodobacter sphaeroides MUTANT LACKING pufX

Abstract— Mutants of Rhodobacler sphac, roicies lacking the pufX gene are unable to grow photosynthetically and have been reported to show impaired cyclic electron flow and elevated B875 levels when grown at low oxygen tension. An examination of the low-temperature optical properties of chromatophores from a pufx- strain, obtained by complementation of a pufLMX mutant in trans with pufQBALM , encoding polypeptides of the B875 antenna and reaction center complexes, revealed that the absorption and fluorescence emission maxima and the fluorescence polarization rise over the B875-Qy band were shifted to lower energies. These results suggest that the pufX gene product may limit the aggregation state of the B875 complex, assuring the proper functional arrangement of antenna and reaction ccntcr components within the cores of the photosynthetic units.     

THE LIGHT-HARVESTING SYSTEM OF THE UNICELLULAR ALGA Mantoniella squamata (PRASINOPHYCEAE): EVIDENCE FOR THE LACK OF A PHOTOSYSTEM I-SPECIFIC ANTENNA COMPLEX

The light-harvesting complexes (LHC) were isolated from the unicellular alga Mantoniella squamata (Prasinophyceae) by sucrose-density centrifugation. Beside the major LHC (II), a photosystem I complex was obtained that could be dissociated into a photosystem I core complex and an associated LHC I. In contrast to other chlorophyll b-containing antennae, both LHC II as well as LHC I were observed to be identical with respect to the following features: the molecular weights, the isoelectric points and the retention behavior on anion-exchange chromatography of the apoproteins, the pigment content and the absorption and fluorescence spectra. We conclude from these results that Mantoniella contains only one homogenous population of LHC, which cooperate with both photosystems not on the basis of specific recognition but on the simple basis of statistical interaction. This is the first report of a chlorophyll b-containing light-harvesting system without any subpopulations: therefore, it is suggested that it arises from a most primitive type of chlorophyll b-containing chloroplast.     

ISOLATION AND CHARACTERIZATION OF A STRUCTURAL SUBUNIT FROM THE CORE LIGHT-HARVESTING COMPLEX OF Rhodobacter sphaeroides 2.4.1 AND puc705-BA

A method for isolating a structural subunit, B825, from the B875 core light-harvesting complex (LHC) of Rhodobacter sphaeroides 2.4.1 (wild-type) and a B800-B850(-) mutant, puc705-BA, is presented. This method, based on one developed to prepare a similar subunit, B820, from the core LHC of Rhodospirillum rubrum [Miller et al., Biochemistry 26, 5055-5062 (1987)], requires the dissociation of treated chromatophores with the detergent, octyl-glucoside. A subsequent gel filtration step separates B800-850 (if present), reaction centers, and free bacteriochlorophyll from the subunit complex. B825 was quantitatively reassociated into an 873 nm absorbing form which resembled the in vivo complex as judged by its absorption properties. The polypeptides in B825 and B800-850 were isolated by HPLC and identified by N-terminal amino acid sequencing. Two polypeptides, alpha and beta, were found in each complex in a 1:1 ratio. The spectral and biochemical properties of the subunits isolated from Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodobacter capsulatus are compared.     

THE EFFECT OF PERIPHERAL SUBSTITUTION ON THE BATHOCHROMIC SHIFT OF THE Qy TRANSITION OF BACTERIOCHLOROPHYLL DIMERS; in vitro MODELS OF THE PROTEIN EFFECT ON THE SPECTRUM OF PIGMENT CENTERS IN THE LIGHT-HARVESTING COMPLEXES*

Abstract— The effect of chemical modifications in the side groups of the isocyclic ring V on the formation, optical absorption and circular dichroism of bacteriochlorophyll (Bchl) dimers was examined in a mixture of formamide and water containing TritonX–100 and variable amounts of pyridine. Substitution of the carbomethoxy group in the C13² position with a hydrogen atom, had no effect on the dimerization constant but increased the shift of the Q_y transition by 1000 cm^-1 with respect to the native Bchl. Substitution of the C13 hydrogen atom with OH decreased the shift of the Q_y transition by 400 cm^-1. The similarity between the spectra of the modified Bchl dimers and Bchl dimers in vivo indicates that protein binding to the side groups at Bchl dimers may profoundly affect the energy of their Q_y transition but have minor effects on the Q_y transitions of the monomelic Bchl.     

SOLVENT EFFECTS ON TRIPLET-STATE BACTERIOCHLOROPHYLL a AS DETECTED BY TRANSIENT RAMAN SPECTROSCOPY AND THE ENVIRONMENT OF BACTERIOCHLOROPHYLL a IN THE LIGHT-HARVESTING COMPLEX OF Rhodobacter sphaeroides R26

The frequency of BChl that was bound to the light-harvesting complex (LHC) of Rhodobacter sphaeroides R26 was found to be 1598 cm^-1, a result which suggests that a pair of BChl molecules form a dimer in the LHC in the T₁ state.     

SITE-DIRECTED MUTAGENESIS OF THE LH2 LIGHT-HARVESTING COMPLEX OF Rhodobacter sphaeroides: CHANGING βLys23 TO Gin RESULTS IN A SHIFT IN THE 850 nm ABSORPTION PEAK

The present study describes the construction of a Rhodobacter sphaeroides light-harvesting (LH2) mutant in which the charged residue βSLys23 is changed by site-directed mutagenesis to a Gin residue, and the characterization of the resulting mutant complex by a range of spectroscopic techniques. In the 77 K absorption spectrum of the mutant, the peak equivalent to the 850 nm peak in the wild-type membrane is blue-shifted by approximately 18 nm to 837 nm; except for this blue-shift, the 77 K. fluorescence excitation and emission spectra and the circular dichroism spectrum of the mutant are very similar to the equivalent spectra from the wild-type membranes, suggesting that the mutation βLys23 → Gin probably does not cause any major changes in the conformation or aggregation state of these membranes. Possible causes of the 18 nm blue-shift in the absorption spectrum are discussed.     

用阳极溶出伏安法测定铅血清蛋白络合物、条件生成常数及血清蛋白的分子量

本文提出利用阳极溶出伏安法测定铅离子与人血清白蛋白,人血清丙种球蛋白和牛血清白蛋白所生成的金属蛋白络合物的组成及其条件生成常数,并测定与铅离子相结合的蛋白质的分子量.     

ON THE SPATIAL CORRELATION BETWEEN THE PROTEIN SURFACE AND THE ELECTRON TRANSFER CYCLE IN BACTERIAL PHOTOSYNTHETIC REACTION CENTERS

Abstract— n -Doxylstearic acids are shown to be electron acceptors in reaction centers from R. spheroides R-26 mutant illuminated with 860 nm light. The electron accepting efficiency varies with n , thus with the location of the nitroxide moiety on the stearic acid chain, and is maximum for 12-doxylstearic acid. In this molecule the nitroxide moiety is 1.4 nm from the polar carboxyl carbon based on molecular models. If the nitroxide moiety is located in or near the polar reaction center protein surface, the results suggest that the quinone-iron complex, which is the ultimate electron acceptor from a bacterio-chlorophyll dimer in the reaction center, is located ∼ 1.4 nm from the protein surface. The protein itself is estimated to have a diameter of 5.4 nm assuming spherical symmetry, so it is postulated that the quinone-iron complex is located on one side of the protein.     

蛋白质定量分析的进展

蛋白质是生命的起源,蛋白的定量分析涉及到生产与科研的各领域和与生命有关的许多行业。文中着重阐述了化述法和免疫法在定量分析蛋白质中的应用及发展动态。     

SHORT-TERM ADAPTATION OF HIGHER PLANTS TO CHANGING LIGHT INTENSITIES AND EVIDENCE FOR THE INVOLVEMENT OF PHOSPHORYLATION OF THE LIGHT HARVESTING CHLOROPHYLL alb PROTEIN COMPLEX OF PHOTOSYSTEM II

Abstract The short-term adaptation of intact leaves to an increase in light intensity was studied by an analysis of chlorophyll fluorescence and oxygen evolution monitored by photoacoustics. An increase in light intensity led to an oxygen “gush”. This “gush” was followed by a large (up to 120%) biphasic increase in the yield of oxygen evolution characterized by a fast phase (T = 0.5–2 min) and a slow phase (T = 4–20 min). The fast phase of the increase in oxygen yield was coupled to a decrease of fluorescence, whereas the slow phase was accompanied by a parallel fluorescence increase. A comparison of fluorescence parameters with oxygen yield indicates that the slow phase of the increase in oxygen yield was coupled to an increase in the antenna size of photosystem II. The slow phase was not inhibited by the uncoupler Nigericin but it was absent in chlorophyll-b-less barley mutants dencient in the light harvesting chlorophyll a/b protein complex of photosystem II (LHC II). These experiments indicate that changes in the LHC II mediated energy distribution, which occur in the time-range of several minutes, are involved in the adaptation to changing light intensities. Moreover, electrophoretic analysis of ³²P orthophosphate labeled leaf discs adapted to low and high light intensities suggests that the slow phase of the increase in oxygen evolution involves dephosphorylation of the 25 kDa polypeptide of LHC II, by a small extent of 12%. The trigger for the slow phase of the increase in oxygen yield does not involve the oxidation of the plastoquinone pool. It was found that in response to the increased light intensity, the plastoquinone pool became more reduced as judged by model calculations. Experiments with the uncoupler Nigericin suggest that the control of the slow phase of adaptation to increased light intensity was also not exerted by the pH gradient across the thylakoid membrane. The similarities between the adaptation to increased light intensity and the state II to state I transition suggest that both adaptation phenomena involve LHC II dephosphorylation possibly triggered by the cytochrome b₆/f complex.     

SHORT-TERM ADAPTATION OF HIGHER PLANTS TO CHANGING LIGHT INTENSITIES AND EVIDENCE FOR THE INVOLVEMENT OF PHOSPHORYLATION OF THE LIGHT HARVESTING CHLOROPHYLL alb PROTEIN COMPLEX OF PHOTOSYSTEM II

Abstract— The short-term adaptation of intact leaves to an increase in light intensity was studied by an analysis of chlorophyll fluorescence and oxygen evolution monitored by photoacoustics. An increase in light intensity led to an oxygen “gush”. This “gush” was followed by a large (up to 120%) biphasic increase in the yield of oxygen evolution characterized by a fast phase (T = 0.5–2 min) and a slow phase (T = 4–20 min). The fast phase of the increase in oxygen yield was coupled to a decrease of fluorescence, whereas the slow phase was accompanied by a parallel fluorescence increase. A comparison of fluorescence parameters with oxygen yield indicates that the slow phase of the increase in oxygen yield was coupled to an increase in the antenna size of photosystem II. The slow phase was not inhibited by the uncoupler Nigericin but it was absent in chlorophyll-b-less barley mutants deñcient in the light harvesting chlorophyll a/b protein complex of photosystem II (LHC II). These experiments indicate that changes in the LHC II mediated energy distribution, which occur in the time-range of several minutes, are involved in the adaptation to changing light intensities. Moreover, electrophoretic analysis of ³²P orthophosphate labeled leaf discs adapted to low and high light intensities suggests that the slow phase of the increase in oxygen evolution involves dephosphorylation of the 25 kDa polypeptide of LHC II, by a small extent of 12%. The trigger for the slow phase of the increase in oxygen yield does not involve the oxidation of the plastoquinone pool. It was found that in response to the increased light intensity, the plastoquinone pool became more reduced as judged by model calculations. Experiments with the uncoupler Nigericin suggest that the control of the slow phase of adaptation to increased light intensity was also not exerted by the pH gradient across the thylakoid membrane. The similarities between the adaptation to increased light intensity and the state II to state I transition suggest that both adaptation phenomena involve LHC II dephosphorylation possibly triggered by the cytochrome b₆/f complex.