紫色光合细菌ThermochromatiumTepidum捕光天线复合物2的激发态动力学

Thermochromatium (Tch.) tepidum是一种中等嗜热的紫色光合细菌, 最佳生长温度为48-50 ℃; 其捕光天线复合物2 (LH2)含有非均一性脱辅基蛋白和类胡萝卜素(Car), 且高分辨率晶体结构未知. 我们通过超快光谱研究了分别采用去垢剂n-dodecyl-β-D-maltoside (DDM)和lauryldimethylamine oxide (LDAO)制备的LH2的激发态动力学, 观测到由细菌叶绿素(BChl)的Qy态介导的B800-to-B850单重态能量传递过程(时间尺度~1.2 ps, 用DDM制备的LH2), 以及由类胡萝卜素S2态介导的Car-to-Car和Car-to-BChl 单重态能量传递过程(~100 fs). 结果表明C=C共轭双键数目(NC=C)为11和12的两类Car共处于同一LH2复合物中; 相对于源自其它菌种、构成组分相对简单的LH2, Tch. tepidum的LH2中B800-B850的相对取向有较大差异. 本工作发现LH2中低含量类胡萝卜素组分anhydrorhodovibrin (NC=C=12)起着高效“能量陷阱”的作用, 可能是一种重要的光保护机制; 基于类胡萝卜素的超快谱带位移现象提出(OH-)spirilloxanthin(NC=C=13)距BChl分子可能比其它类胡萝卜素更近. 这些研究结果有助于进一步理解苛刻自然条件下生长的Tch. tepidum的捕光和光保护机制.     

水杨酸-2’-乙基己基酯在胶束中的增溶位点

考察了阳离子、非离子和阴离子表面活性剂存在下水杨酸-2’-乙基己基酯(EHS)的吸收光谱和激发态分子内质子转移(ESIPT)荧光光谱. 结果表明, EHS可增溶在胶束中, 2’-乙基己基碳链朝向胶束内核, 而水杨酸基朝向胶束-水界面; 胶束环境有利于EHS分子对紫外光的吸收和分子内氢键的形成, 从而使ESIPT 荧光显著增强, 激发态分子以发射可见光和非辐射去活化方式衰减; 并根据EHS和表面活性剂分子的结构和大小, 解释了EHS分子在胶束中的结合位点, 荧光猝灭和酯水解的光谱测量进一步为此结合位点提供了佐证.     

水杨酸-2′-乙基己基酯在胶束中的增溶位点

考察了阳离子、非离子和阴离子表面活性剂存在下水杨酸-2′-乙基己基酯(EHS)的吸收光谱和激发态分子内质子转移(ESIPT)荧光光谱.结果表明,EHS可增溶在胶束中,2′-乙基己基碳链朝向胶束内核,而水杨酸基朝向胶束-水界面;胶柬环境有利于EHS分子对紫外光的吸收和分子内氢键的形成,从而使ESIPT荧光显著增强,激发态分子以发射可见光和非辐射去活化方式衰减;并根据EHS和表面活性剂分子的结构和大小,解释了EHS分子在胶束中的结合位点,荧光猝灭和酯水解的光谱测量进一步为此结合位点提供了佐证.     

正负离子和表面活性剂对水解聚丙烯酰胺分子线团尺寸的影响及其作用机理

采用动态光散射(DLS)方法,研究了无机电解质正离子与负离子对部分水解聚丙烯酰胺(HPAM)分子线团尺寸的影响,也研究了阴离子型表面活性剂与非离子型表面活性剂对HPAM分子线团尺寸的影响.结果表明,无机电解质负离子对HPAM分子尺寸(分子流体力学直径(Dh))影响较小,而无机电解质正离子对Dh的影响较大,且影响程度随正离子浓度增大而减小.Ca2+、Mg2+、K+和Na+对Dh的作用强弱顺序为Mg2+>Ca2+>Na+>K+.当向聚合物溶液中加入阴离子型表面活性剂时,随表面活性剂浓度增大,Dh先减小,后增大,再减小.此外,由于强烈的静电斥力作用,阴离子型表面活性剂分子在聚合物分子表面吸附较弱,难形成"表面活性剂-聚合物"络合物,而非离子型表面活性剂会以类似于胶束聚集体的形式吸附在聚合物分子链上,形成"表面活性剂-聚合物"络合物,结果造成Dh随表面活性剂浓度增加而逐渐增大.     

分子动力学模拟研究荧光分子芘在磺基甜菜碱胶束中的增溶

采用分子动力学模拟研究了荧光分子芘在磺基甜菜碱两性表面活性剂聚集体中的增溶现象．结果表明,芘分子自发地自溶液中增溶进入胶束疏水内核的栅栏层区域．当胶束溶液中芘分子的局部浓度增大时,两个芘分子可以同时增溶进胶束的栅栏层区域,此时两个芘分子形成π-π共轭堆积的激发态络合物．但是由于荧光分子之间的弱兀．兀相互作用,激发态络合物在胶束中是不稳定的,表现为两个芘分子的多次结合和分离．模拟表明,分子动力学方法可以在分子水平上研究荧光探针分子在表面活性剂胶束中的增溶位点,解释荧光分子在胶束中的动力学现象．     

新型Gemini咪唑表面活性剂的合成及表面性能

以2,2-双(溴甲基)-1,3-丙二醇为连接基合成了新型的连接基为枝状的Gemini咪唑表面活性剂2,4-二(溴化-3-烷基咪唑)-1,3-丙二醇([Cn-P-Cnim]Br2,n=10,12,14).产物经核磁共振氢谱(1H NMR)、红外(IR)光谱和元素分析等进行了分析,证明所得产物即为目标产物.通过表面张力法和电导法测量其表面活性并计算胶束形成热力学参数(ΔG m—0,ΔH m—0,ΔS m—0).结果表明,25℃时3种表面活性剂均具有很高的表面活性,胶束的形成是自发的熵驱动过程.     

阴离子表面活性剂与阳离子的相互作用

用密度泛函理论, 在B3LYP/6-31G水平上, 对十二烷基磺酸盐和羧酸盐阴离子表面活性剂与阳离子(Na+, Ca2+, Mg2+)形成的离子对进行结构优化, 从分子水平上研究表面活性剂与阳离子之间的相互作用. 计算结果表明: 磺酸盐和羧酸盐表面活性剂均采用2:1型, 即极性头中两个氧原子与阳离子发生稳定结合; 在与阳离子结合之前, 表面活性剂分子上的α-亚甲基带有明显的负电荷, 因此将其归为极性头; 但在阳离子电荷诱导下, α-亚甲基转而带有部分弱正电荷, 使极性头范围缩小. 计算也发现, 表面活性剂尾链带有弱正电荷, 使胶束内核带有了部分极性, 利于表面活性剂在溶液中的聚集, 此种极性介于烷烃油相和水相的极性之间.     

转基因抗虫蛋白Cry1Ac与表面活性剂作用的芘探针光谱分析

以芘作为外源荧光探针,采用牛血清蛋白作为参照,考察了转基因抗虫蛋白Cry1Ac与4种表面活性剂相互作用的荧光光谱特征.结果表明,鼠李糖脂和Tween 80体系的芘荧光行为有相似的变化规律,可与蛋白质发生稳定的缔合过程.Cry1Ac蛋白的亲水性和分子极性较强,在表面活性剂浓度低时,可导致芘的I1/I3值较高.在阴离子、非离子表面活性剂胶团形成后,2种蛋白质的存在均不改变芘与表面活性剂的结合位点.十六烷基三甲基溴化铵对芘的荧光有一定猝灭作用,且在Cry1Ac蛋白介入下不能形成稳定的胶束.     

季铵盐型Gemini表面活性剂的胶束化动力学研究

采用停流法并结合Aniannson-Wall理论, 研究了联接基为(CH2)2, (CH2)3, (CH2)4和(CH2)6的季铵盐型Gemini表面活性剂胶束的形成-破坏过程. 动力学的研究结果表明, 胶束形成-破坏过程的弛豫时间(τ2)与联接基的长度、表面活性剂的浓度、反离子的浓度以及温度有关. 随联接基长度的增加, 季铵盐型Gemini表面活性剂胶束形成-破坏过程的弛豫时间缩短. 当温度高于293 K时, 随着反离子浓度的增加, 1/τ2将出现一个最低值. 根据核化焓结果提出了不同的联接基长度的季铵盐型Gemini表面活性剂具有不同的胶束形成-破坏过程的机理.     

DMABN在表面活性剂胶束水溶液中的荧光性质

研究了对二甲氨基苯甲腈(DMABN)在各种胶束水溶液中的荧光光谱性质, 发现不同胶束栅栏层区域的不同性质影响了探针的分子内扭转电荷转移(TICT)特性. 对离子型胶束, 头基电场是主要影响因素, 促进了DMABN分子TICT态的形成, 反离子解离度琢越大, 胶束溶液中的Ia/Ib越强. 在非离子表面活性剂胶束中, 聚氧乙烯链环外壳包裹的大量水使其氢键影响明显, 而很短的聚氧乙烯链还可能带来端基氢的氢键作用. 从DMABN的光物理特性看, 欲将胶束作为分散载体利用其TICT态特性, 选择反离子解离度较大的阴离子胶束(例如SDS或SDSO)较好.     

Selective chemical shift assignment of B800 and B850 bacteriochlorophylls in uniformly [13C,15N]-labeled light-harvesting complexes by solid-state NMR spectroscopy at ultra-high magnetic field

The electronic ground states of the bacteriochlorophyll a type B800 and type B850 in the light-harvesting 2 complex of Rhodopseudomonas acidophila strain 10050 have been characterized by magic angle spinning (MAS) dipolar (13)C-(13)C correlation NMR spectroscopy. Uniformly [(13)C,(15)N] enriched light-harvesting 2 (LH2) complexes were prepared biosynthetically, while [(13)C,(15)N]-B800 LH2 complexes were obtained after reconstitution of apoprotein with uniformly [(13)C,(15)N]-enriched bacteriochlorophyll cofactors. Extensive sets of isotropic (13)C NMR chemical shifts were obtained for each bacteriochlorin ring species in the LH2 protein. (13)C isotropic shifts in the protein have been compared to the corresponding shifts of monomeric BChl a dissolved in acetone-d(6). Density functional theory calculations were performed to estimate ring current effects induced by adjacent cofactors. By correction for the ring current shifts, the (13)C shift effects due to the interactions with the protein matrix were resolved. The chemical shift changes provide a clear evidence for a global electronic effect on the B800 and B850 macrocycles, which is attributed to the dielectrics of the protein environment, in contrast with local effects due to interaction with specific amino acid residues. Considerable shifts of -6.2 < Deltasigma < +5.8 ppm are detected for (13)C nuclei in both the B800 and the B850 bacteriochlorin rings. Because the shift effects for the B800 and B850 are similar, the polarization of the electronic ground states induced by the protein environment is comparable for both cofactors and corresponds with a red shift of approximately 30 nm relative to the monomeric BChl dissolved in acetone-d(6). The electronic coupling between the B850 cofactors due to macrocycle overlap is the predominant mechanism behind the additional red shift in the B850.     

Reconstituted LH2 in multilayer membranes induced by poly-l-lysine: Structure of supramolecular and electronic states

To explore the effect of cell membrane stacking on the function of light-harvesting complex 2 (LH2) in purple nonsulfur photosynthetic bacteria, LH2 from Rhodobacter sphaeroides 2.4.1 (R. sph 2.4.1) was reconstituted into lipid bilayer vesicles (LH2@liposome) and further formed multi-layer structure by electrostatic interaction with poly-l-lysine (LH2@liposome/PLL), which was characterized by cryo-electron microscopy (cryo-EM) and TEM. When embedded in liposomes and additionally in multi-layer liposomes, the absorption band, zero-crossing point of CD signals and fluorescence emission of LH2 B850 excitons were observed to uniformly have 1–2 nm red-shifting. Combining with the corresponding fluorescence quench and the generation of shorter-living fluorescence species, a new excitonic species generated through B850 structural splitting was proposed. By FT-Raman and triplet carotenoid dynamics, the structural mechanism was deduced and discussed. Briefly, all environmental changes, including LH2 aggregating and multi-layer membrane stacking, eventually applied forces on B850 exciton molecules mainly through the hydrogen bonding between the C3-acetyl carbonyl groups of B850 BChls and Tyr44 and 45 residues at C-terminus of α-polypeptides. The effect of multi-layer structure on LH2 could be assigned as a kind of photoprotection.     

Carotenoids Do Not Protect Bacteriochlorophylls in Isolated Light-Harvesting LH2 Complexes of Photosynthetic Bacteria from Destructive Interactions with Singlet Oxygen

The effect of singlet oxygen on light-harvesting (LH) complexes has been studied for a number of sulfur (S⁺) and nonsulfur (S⁻) photosynthetic bacteria. The visible/near-IR absorption spectra of the standard LH2 complexes (B800-850) of Allochromatium (Alc.) vinosum (S⁺), Rhodobacter (Rba.) sphaeroides (S⁻), Rhodoblastus (Rbl.) acidophilus (S⁻), and Rhodopseudomonas (Rps.) palustris (S⁻), two types LH2/LH3 (B800-850 and B800-830) of Thiorhodospira (T.) sibirica (S⁺), and an unusual LH2 complex (B800-827) of Marichromatium (Mch.) purpuratum (S⁺) or the LH1 complex from Rhodospirillum (Rsp.) rubrum (S⁻) were measured in aqueous buffer suspensions in the presence of singlet oxygen generated by the illumination of the dye Rose Bengal (RB). The content of carotenoids in the samples was determined using HPLC analysis. The LH2 complex of Alc. vinosum and T. sibirica with a reduced content of carotenoids was obtained from cells grown in the presence of diphenylamine (DPA), and LH complexes were obtained from the carotenoidless mutant of Rba. sphaeroides R26.1 and Rps. rubrum G9. We found that LH2 complexes containing a complete set of carotenoids were quite resistant to the destructive action of singlet oxygen in the case of Rba. sphaeroides and Mch. purpuratum. Complexes of other bacteria were much less stable, which can be judged by a strong irreversible decrease in the bacteriochlorophyll (BChl) absorption bands (at 850 or 830 nm, respectively) for sulfur bacteria and absorption bands (at 850 and 800 nm) for nonsulfur bacteria. Simultaneously, we observe the appearance of the oxidized product 3-acetyl-chlorophyll (AcChl) absorbing near 700 nm. Moreover, a decrease in the amount of carotenoids enhanced the spectral stability to the action of singlet oxygen of the LH2 and LH3 complexes from sulfur bacteria and kept it at the same level as in the control samples for carotenoidless mutants of nonsulfur bacteria. These results are discussed in terms of the current hypothesis on the protective functions of carotenoids in bacterial photosynthesis. We suggest that the ability of carotenoids to quench singlet oxygen (well-established in vitro) is not well realized in photosynthetic bacteria. We compared the oxidation of BChl850 in LH2 complexes of sulfur bacteria under the action of singlet oxygen (in the presence of 50 μM RB) or blue light absorbed by carotenoids. These processes are very similar: {[BChl + (RB or carotenoid) + light] + O₂} → AcChl. We speculate that carotenoids are capable of generating singlet oxygen when illuminated. The mechanism of this process is not yet clear.     

Structure-Based Calculations of the Optical Spectra of the LH2 Bacteriochlorophyll-Protein Complex from Rhodopseudomonas acidophila

Abstract— The molecular structure of the light-harvesting complex 2 (LH2) bacteriochlorophyll-protein antenna complex from the purple non-sulfur photosynthetic bacterium Rhodopseudomonas acidophila , strain 10050 provides the positions and orientations of the 27 bacteriochlorophyll (BChl) molecules in the complex. Our structure-based model calculations of the distinctive optical properties (absorption, CD, polarization) of LH2 in the near-infrared region use a point-monopole approximation to represent the BChl Q_y transition moment. The results of the calculations support the assignment of the ring of 18 closely coupled BChl to B850 (BChl absorbing at 850 nm) and the larger diameter, parallel ring of 9 weakly coupled BChl to B800. All of the significantly allowed transitions in the near infrared are calculated to be perpendicular to the C9 symmetry axis, in agreement with polarization studies of this membrane-associated complex. To match the absorption maxima of the B800 and B850 components using a relative permittivity (dielectric constant) of 2.1, we assign different site energies (12 500 and 12260 cm⁻¹, respectively) for the Q_y transitions of the respective BChl in their protein binding sites. Excitonic coupling is particularly strong among the set of B850 chromophores, with pairwise interaction energies nearly 300 cm between nearest neighbors, comparable with the experimental absorption bandwidths at room temperature. These strong interactions, for the full set of 18 B850 chromophores, result in an excitonic manifold that is 1200 cm⁻¹ wide. Some of the upper excitonic states should result in weak absorption and perhaps stronger CD features. These predictions from the calculations await experimental verification.     

Ultrafast excitation relaxation in light-harvesting complex LH2 from Rb.sphaeroides 601

The light-driven reactions of photosynthesis are the means by which nature converts solar energy into electrochemical potential, which is eventually stored as chemical energy. These initial reactions occur in two closely coupled pigment systems, the network of so-called antenna system in which the excitation en-ergy is absorbed by the pigments and efficiently transported to another system, the photosynthetic reac-tion center where the energy is converted into a stable trans-membrane charge sepa…     

Ultrafast excitation relaxation in light-harvesting complex LH2 from <Emphasis Type="Italic">Rb. sphaeroides</Emphasis> 601

The energy relaxation and kinetic evolution of transient spectra of bacteriochlorophylls (BChls) in light-harvesting complex LH2 from Rb. sphaeroides 601 were investigated using femtosecond pump-probe technique. Upon 783 nm excitation, the energy at B800 BChls experiences an intramolecular redistribution with 0.35 ps time constant before transferring to B850 BChls. With tuning the excitation wavelength, the dynamical evolution of excited BChls was clearly observed, which indicates an obvious competition between the ground state bleaching and excited state absorption (ESA) of BChls involved and an isosbestic point near 818 nm, and also demonstrates that from the lower electronic excited state of B800 BChls to the higher excitonic state of B850 BChls is an efficient routine for energy transfer. The excitation energy in higher excitonic states of B850 BChls relaxes rapidly to the next lowest excitonic state by interconversion, delocalization to adjacent molecular, populating the lowest excitonic state and the change of molecular conformation.     

Spectral Changes Induced by Alkaline pH and Specific Chemical Modification of Amino Acid Residues in the Light-Harvesting II Antenna Complex from Ectothiorhodospira sp.

Abstract— The absorption spectrum of the membrane-bound light-harvesting (LH)II antenna complex from Ectothiorhodospira sp. has two characteristic near-infrared bands at 797 (B800 band) and 857 (B850 band) nm. Alkaline pH induced a B850 band blue shift of 17–21 nm depending on experimental conditions. The blue shift was totally reversible when the original experimental conditions were re-established. No significant effect was observed, however, on the B800 band under the same experimental conditions. The intensity and shape of the pigment circular dichroism signals were maintained with the exception of a blue shift of the signal from the B850 band concomitant with the blue shift of that absorption band. Specific chemical modification of the LHII complex with salicylaldehyde allowed correlation of the alkaline pH effect with the neutralization of a lysine positive charge. We propose that the observed blue shift of the B850 band is due to distortion of the bacteriochlorophyll domain as a consequence of electrostatic and probably hydrogen-bonding changes but not due to modification of the pigment excitonic interactions within the pigment-protein complex.     

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.     

On the binding of calcium by micelles composed of carboxy-modified pluronics measured by means of differential potentiometric titration and modeled with a self-consistent-field theory

We perform differential potentiometric titration measurements for the binding of Ca2+ ions to micelles composed of the carboxylic acid end-standing Pluronic P85 block copolymer (i.e., CAE-85 (COOH-(EO)26-(PO)39-(EO)26-COOH)). Two different ion-selective electrodes (ISEs) are used to detect the free calcium concentration; the first ISE is an indicator electrode, and the second is a reference electrode. The titration is done by adding the block copolymers to a known solution of Ca2+ at neutral pH and high enough temperature (above the critical micellization temperature CMT) and various amount of added monovalent salt. By measuring the difference in the electromotive force between the two ISEs, the amount of Ca2+ that is bound by the micelles is calculated. This is then used to determine the binding constant of Ca2+ with the micelles, which is a missing parameter needed to perform molecular realistic self-consistent-field (SCF) calculations. It turns out that the micelles from block copolymer CAE-85 bind Ca2+ ions both electrostatically and specifically. The specific binding between Ca2+ and carboxylic groups in the corona of the micelles is modeled through the reaction equilibrium -COOCa+ <==> -COO- + Ca2+ with pKCa = 1.7 +/- 0.06.     

Ultrahigh field MAS NMR dipolar correlation spectroscopy of the histidine residues in light-harvesting complex II from photosynthetic bacteria reveals partial internal charge transfer in the B850/His complex.

Low-temperature 15N and 13C CP/MAS (cross-polarization/magic angle spinning) NMR has been used to analyze BChl-histidine interactions and the electronic structure of histidine residues in the light-harvesting complex II (LH2) of Rhodopseudomonas acidophila. The histidines were selectively labeled at both or one of the two nitrogen sites of the imidazole ring. The resonances of histidine nitrogens that are interacting with B850 BChl a have been assigned. Specific 15N labeling confirmed that it is the tau-nitrogen of histidines which is ligated to Mg2+ of B850 BChl molecules (beta-His30, alpha-His31). The pi-nitrogens of these Mg2+-bound histidines were found to be protonated and may be involved in hydrogen bond interactions. Comparison of the 2-D MAS NMR homonuclear (13C-13C) dipolar correlation spectrum of [13C6,15N3]-histidines in the LH2 complex with model systems in the solid state reveals two different classes of electronic structures from the histidines in the LH2. In terms of the 13C isotropic shifts, one corresponds to the neutral form of histidine and the other resembles a positively charged histidine species. 15N-13C double-CP/MAS NMR data provide evidence that the electronic structure of the histidines in the neutral BChl a/His complexes resembles the positive charge character form. While the Mg...15N isotropic shift confirms a partial positive charge transfer, its anisotropy is essentially of the lone pair type. This provides evidence that the hybridization structure corresponding to the neutral form of the imidazole is capable of "buffering" a significant amount of positive charge.