Protein identification of purified particles isolated from spinach thylakoids by deoxycholate electrophoresis

Three thylakoid complexes were isolated by deoxycholate preparative electrophoresis. The protein composition of each fraction was analyzed by SDS analytical electrophoresis. No protein of the PS 1 enriched fraction (fraction 1) was found in the PS 2 enriched fraction (fraction 2) and inversely. The antenna complex (fraction 3) did not have any contamination by proteins of fraction 1 or fraction 2. Fraction 1 was mainly composed of the CP1, the reaction center complex of the PS1, and by low molecular weight proteins, previously found in other PS 1 preparations. Tentative assignments of these proteins are presented; among them are iron sulfur proteins. After analytical SDS electrophoresis of fraction 2, the reaction center complex was dissociated. Nevertheless three proteins of 50 kD, 42 kD and 35 kD were assigned to this complex. Fraction 2 contained also the three cytochromes of the thylakoid membranes: cyt f, cyt b6, cyt b559. Fraction 3 was exclusively composed of one protein pigment complex, CP2.Abbreviations SDS sodium dodecyl sulfate - PS 1 photosystem 1 - PS 2 photosystem 2 - CP1, CP2 protein pigment complexes isolated by SDS electrophoresis - cyt cytochromes - P700 primary electron donor of PS 1 - P680 primary electron donor of PS 2 - DOC deoxycholate - Q primary plastoquinone electron acceptor - CF coupling factor     

Crystallization of the reaction center from Rhodobacter sphaeroides in a new tetragonal form

The reaction center from the nonsulfur purple bacteriumRhodobacter sphaeroideshas been crystallized in a new form. The crystals grew in the presence of polyethylene glycol 4000, the detergent β-octyl glucoside, and the amphiphiles heptane triol and benzamidine hydrochloride, using the sitting drop method. The space group of these crystals is tetragonal, P4₁(4₃)2₁2, and the cell constants are a = b = 141.5 Å and c = 276.7 Å with probably 2 proteins per asymmetric unit. A native data set has been set collected to a resolution of 2.8 Å consisting of 56,332 unique reflections (50,731 with F > 2σ) with anR_sym of 9.5%. Analysis of the diffraction data is underway using molecular and isomorphous replacement. © 1994 Wiley-Liss, Inc.     

GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern

We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.     

Engineering stabilising beta-sheet interactions into a conformationally flexible region of the folding transition state of ubiquitin

Protein engineering studies suggest that the transition state for the folding of ubiquitin is highly polarised towards the N-terminal part of the sequence and involves a nucleus of residues within the beta-hairpin (residues 1-17) and main alpha-helix (residues 23-34). In contrast, the observation of small phi-values for residues in the C-terminal portion of the sequence (residues 35-76), coupled with a folding topology that results in a much higher contact order, suggests that fast folding of ubiquitin is dependent upon configurational flexibility in the C-terminal part of the polypeptide chain to ensure passage down a relatively smooth folding funnel to the native state. We show that the introduction of a small mini-hairpin motif as an extension of the native 43-50 hairpin stabilises local interactions in the C-terminal part of the sequence, resulting largely in a deceleration of the unfolding kinetics without perturbing the apparent two-state folding mechanism. However, a single-point Leu-->Phe substitution within the engineered hairpin sequence leads to the premature collapse of the denatured ensemble through the stabilisation of non-native interactions and the population of a compact intermediate. Non-linear effects in the kinetic data at low concentrations of denaturant suggest that the collapsed state, which is further stabilised in the presence of cosmotropic salts, may subsequently fold directly to the native state through a "triangular" reaction scheme involving internal rearrangement rather than unfolding and refolding.     

Temperature-induced complementarity as a mechanism for biomolecular assembly

Recent advances in the measurement and theory of “hydration” interactions between biomolecules provide a basis on which to formulate mechanisms of biomolecular recognition. In this paper we have developed a mathematical formalism for analyzing specificity encoded in dynamic distributions of surface polar groups, a formalism that incorporates newly recognized properties of directly measured “hydration” forces. As expected, attraction between surfaces requires complementary patterns of surface polar groups. In contrast to usual expectations, thermal motion can create these complementary surface configurations. We have demonstrated that assembly can occur with an increase in conformational entropy of polar residues. Elevated temperature then facilitates recognition rather than hinders it. This mechanism might underlie some temperature-favored assembly reactions common in biological systems that are usually associated with the “hydrophobic effect” only. © 1994 Wiley-Liss, Inc.     

Pitt,a Novel Tetratricopeptide Repeat Protein Involved in Light-Dependent Chlorophyll Biosynthesis and Thylakoid Membrane Biogenesis in Synechocystis sp. PCC 6803

Biogenesis of photosynthetic pigment/protein complexes is a highly regulated process that requires various assisting factors. Here, we report on the molecular analysis of the Pitt gene （sir1644） from the cyanobacterium Synechocystis sp. PCC 6803 （Synechocystis 6803） that encodes a membrane-bound tetratricopeptide repeat （TPR） protein of formerly unknown function. Targeted inactivation of Pitt affected photosynthetic performance and light-dependent chlorophyll synthesis. Yeast two-hybrid analyses and native PAGE strongly suggest a complex formation between Pitt and the light-dependent protochlorophyllide oxidoreductase （POR）. Consistently, POR levels are approximately threefold reduced in the pitt insertion mutant. The membrane sublocalization of Pitt was found to be dependent on the presence of the periplasmic photosystem Ⅱ （PSⅡ） biogenesis factor PratA, supporting the idea that Pitt is involved in the early steps of photosynthetic pigment/protein complex formation.     

一种分析叶绿体类囊体膜色素蛋白复合物的蓝绿温和胶电泳系统

采用蓝绿温和胶电泳系统可以非常有效地分离叶绿体蛋白质复合物,包括PSⅠ, PSⅡ, ATP合酶,细胞色素b₆f复合物,捕光色素复合物和1,5-二磷酸核酮糖羧化酶.还结合SDS-聚丙烯酰胺凝胶电泳将叶绿体多亚基复合物的50多种蛋白质分开,利用免疫印迹对蛋白质复合物进行了初步鉴定,同时还应用蓝色温和胶电泳分析基质、基粒类囊体复合物的组成.     

Chlorophyll fluorescence changes at high temperatures induced by linear heating of greening barley leaves

A relative decrease of the high temperature part (above 60°C) of the chlorophyll fluorescence temperature curve during 3 h to 10 h greening period of barley (Hordeum vulgare L.) leaves was found to be concomitant to a decrease of Chl alb ratio and to a gradual increase of LHCP/core ratio found by electrophoresis and the ratio of granal to total length of thylakoid membranes. It is suggested that the high temperature part of the fluorescence temperature curve depends inversely on the relative amount of LHC II in thylakoid membranes.Abbreviations Chl a(b) chlorophyll a(b) - CPa chlorophyll a protein complex of PS II - CP1 P700 chlorophyll a protein complex of PS I - FP free pigments - FTC fluorescence temperature curve - F(T30) fluorescence intensity at 30°C - LHC II light harvesting complex II - LHCP light harvesting chlorophyll protein - LHCP3 (LHCPm) monomeric form of LHC II - LHCPo oligomeric form of LHC II complex - M1 first maximum of FTC - M2 second maximum (region) of FTC - PAA polyacrylamide - PAR photosynthetically active radiation - PS I(II) Photosystem I(II) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Photosynthetic organisms and excess of metals

When cells get metals in small excess, mechanisms of avoidance occur, such as exclusion, sequestration, or compartmentation. When the excess reaches sub-lethal concentrations, the oxidative stress, that toxic metals trigger, leads to persistent active oxygen species. Biomolecules are then destroyed and metabolism is highly disturbed. At the chloroplast level, changes in pigment content and lipid peroxidation are observed. The disorganized thylakoids impair the photosynthetic efficiency. The Calvin cycle is also less efficient and the photosynthetic organism grows slowly. When an essential metal is given together with a harmful one, the damages are less severe than with the toxic element alone. Combined metals and phytochelatins may act against metal toxicity.     

低温强光下籼粳稻紫黄质脱环氧化酶活性和类囊体膜脂不饱和度的变化

为了阐明籼稻(oryza sativa L.spp.indica)、粳稻(O.sativa L.spp.japonica)对低温强光敏感性的差异,着重研究了低温强光下水稻类囊体膜脂不饱和度与叶黄素循环的变化.随着低温强光处理时间的延长,类囊体膜脂不饱和脂肪酸含量降低,饱和脂肪酸含量增加,因而膜脂不饱和指数(IUFA)下降.同时,叶黄素循环的关键酶--紫黄质脱环氧化酶(VDE)活性降低,叶黄素循环组分中紫黄质(V)含量增加,而单环氧玉米黄质(A)和下米黄质(Z)的含量减少,表现为(A+Z)/(A+Z+V)比值下降.Arrhenius分析证明,VDE对低温和膜脂不饱和度都敏感.相关分析表明,类囊体IUFA分别与VDE活性、(A+Z)/(A +Z+V)和D1蛋白量呈显著的正相关.与粳稻9516相比,籼稻油优63类囊体膜的IUFA较低,低温下类囊体膜脂流动性和稳定性较差,VDE活性和(A+Z)/(A+Z+V)比值较低.     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.     

色素分子在光合膜内取向初探

从正常大麦与缺乏叶绿素b大麦突变种的类囊体中分离提纯捕光叶绿素a /b—蛋白复合体和光系统Ⅱ颗粒,比较其线二色光谱,以及光合膜受到不饱和脂肪酸处理时线二色光谱的变化,参照几种光合色素的标准吸收光谱,初步分析和探讨了叶绿体光合膜中各种线二色组分可能的取向,提出了难以分辩的710～730 nm区域、624nm和595 nm处存在弱二色成分的证据。 七种高等植物叶绿体线二色光谱的成分大致相同。离体叶绿体在不同贮存条件(温度、时间等)下的线二色光谱,在一定范围内表明其色素及色素蛋白复合体具有稳定性。     

低温强光下籼粳稻紫黄质脱环氧化酶活性和类囊体膜脂不饱和度的变化

为了阐明籼稻(Oryza sativa L.spp.indica)、粳稻(O.sativa L.spp.japonica)对低温强光敏感件的差异,着重研究了低温强光下水稻类囊体膜脂不饱和度与叶黄素循环的变化。随着低温强光处理时间的延长,类囊体膜脂不饱和脂肪酸含量降低,饱和脂肪酸含量增加,因而膜脂不饱和指数(IUFA)下降。同时,叶黄素循环的关键酶——紫黄质脱环氧化酶(VDE)活性降低,叶黄素循环组分中紫黄质(V)含量增加,而单环氧玉米黄质(A)和玉米黄质(Z)的含量减少,表现为(A Z)／(A Z V)比值下降。Arrhenius分析证明,VDE对低温和膜脂不饱和度都敏感。相关分析表明,类囊体IUFA分别与VDE活性、(A Z)／(A Z V)和D1蛋白量呈显著的正相关。与粳稻9516相比,籼稻汕优63类囊体膜的IUFA较低,低温下类囊体膜脂流动性和稳定性较筹,VDE活性和(A Z)／(A Z V)比值较低。     

Association of neighboring β-strands of outer membrane protein A in lipid bilayers revealed by site-directed fluorescence quenching

We present a detailed study on the formation of neighboring β-strands during the folding of a monomeric integral membrane protein of the β-barrel type. β-Strand and β-barrel formations were investigated for the eight-stranded transmembrane domain of outer membrane protein A (OmpA) with single-tryptophan (W), single-cysteine (C) OmpA mutants. Based on the OmpA structure, W and C were introduced in two neighboring β-strands oriented toward the hydrocarbon core of the membrane. Replaced residue pairs were closer to either the periplasmic turns (named cis-side) or the outer loops (named trans-side) of the strand. W_nC_m OmpA mutants containing W at position n and C at position m along the polypeptide chain were labeled at the C by a nitroxyl spin label, which is a short-range fluorescence quencher. To monitor the association of neighboring β-strands, we determined the proximity between fluorescent W and labeled C in OmpA folding experiments by intramolecular fluorescence quenching. Formation of native β-strand contacts in folding experiments required the lipid membrane. Residues in the trans-side of strands β₁, β₂, and β₃, represented by mutants W₁₅C₃₅ (β₁β₂, trans) and W₅₇C₃₅ (β₃β₂, trans), reached close proximity prior to residues in the N(β₁)- and C(β₈)-terminal strands as examined for mutants W₁₅C₁₆₂ (β₁β₈, trans) and W₇C₁₇₀ (β₁β₈, cis). Tryptophan and cysteine converged slightly faster in W₁₅C₁₆₂ (β₁β₈, trans) than in W₇C₁₇₀ (β₁β₈, cis). The last folding step was observed for residues at the cis-ends of strands β₁ and β₂ for the mutant W₇C₄₃ (β₁β₂, cis). The data also demonstrate that the neighboring β-strands associate upon insertion into the hydrophobic core of the lipid bilayer.     

Heme binding properties of heterologously expressed spinach cytochrome b(6): implications for transmembrane b-type cytochrome formation

In vivo and in vitro requirements for the formation of cytochrome b(6) were examined to analyze the mechanisms of transmembrane b-type cytochrome formation. After heterologous expression of spinach cytochrome b(6), formation of the holo-cytochrome was observed within the E. coli inner membrane. The transmembrane orientation of cytochrome b(6) appeared not to be critical for heme binding and holo-cytochrome formation. Furthermore, in vitro reconstitution of cytochrome b(6) was possible under oxidizing as well as under reducing conditions. Taken together these observations strongly indicate that transmembrane b-type cytochromes can spontaneously assemble in vitro as well as in a membrane.