Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin

The transport of protons across membranes is an important process in cellular bioenergetics. The light-driven proton pump bacteriorhodopsin is the best-characterized protein providing this function. Photon energy is absorbed by the chromophore retinal, covalently bound to Lys 216 via a protonated Schiff base. The light-induced all-trans to 13-cis isomerization of the retinal results in deprotonation of the Schiff base followed by alterations in protonatable groups within bacteriorhodopsin. The changed force field induces changes, even in the tertiary structure, which are necessary for proton pumping. The recent report of a high-resolution X-ray crystal structure for the late M intermediate of a mutant bacteriorhopsin (with Asp 96-->Asn) displays the structure of a proton pathway highly disturbed by the mutation. To observe an unperturbed proton pathway, we determined the structure of the late M intermediate of wild-type bacteriorhodopsin (2.25 A resolution). The cytoplasmic side of our M2 structure shows a water net that allows proton transfer from the proton donor group Asp 96 towards the Schiff base. An enlarged cavity system above Asp 96 is observed, which facilitates the de- and reprotonation of this group by fluctuating water molecules in the last part of the cycle.     

Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin

A wide variety of mechanisms are used to generate a proton-motive potential across cell membranes, a function lying at the heart of bioenergetics. Bacteriorhodopsin, the simplest known proton pump, provides a paradigm for understanding this process. Here we report, at 2.1 A resolution, the structural changes in bacteriorhodopsin immediately preceding the primary proton transfer event in its photocycle. The early structural rearrangements propagate from the protein's core towards the extracellular surface, disrupting the network of hydrogen-bonded water molecules that stabilizes helix C in the ground state. Concomitantly, a bend of this helix enables the negatively charged primary proton acceptor, Asp 85, to approach closer to the positively charged primary proton donor, the Schiff base. The primary proton transfer event would then neutralize these two groups, cancelling their electrostatic attraction and facilitating a relaxation of helix C to a less strained geometry. Reprotonation of the Schiff base by Asp 85 would thereby be impeded, ensuring vectorial proton transport. Structural rearrangements also occur near the protein's surface, aiding proton release to the extracellular medium.     

Crystal structure of metarhodopsin II

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta?II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta?II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta?II, presuming that the crystal's high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0?? and 2.85?? crystal structures, respectively, of Meta?II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta?II structures, the electron density from the retinal ligand seamlessly continues into the Lys?296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta?II. The structures can now serve as models for the large GPCR family.     

Molecular mechanism of vectorial proton translocation by bacteriorhodopsin

Bacteriorhodopsin, a membrane protein with a relative molecular mass of 27,000, is a light driven pump which transports protons across the cell membrane of the halophilic organism Halobacterium salinarum. The chromophore retinal is covalently attached to the protein via a protonated Schiff base. Upon illumination, retinal is isomerized. The Schiff base then releases a proton to the extracellular medium, and is subsequently reprotonated from the cytoplasm. An atomic model for bacteriorhodopsin was first determined by Henderson et al, and has been confirmed and extended by work in a number of laboratories in the last few years. Here we present an atomic model for structural changes involved in the vectorial, light-driven transport of protons by bacteriorhodopsin. A 'switch' mechanism ensures the vectorial nature of pumping. First, retinal unbends, triggered by loss of the Schiff base proton, and second, a protein conformational change occurs. This conformational change, which we have determined by electron crystallography at atomic (3.2 A in-plane and 3.6 A vertical) resolution, is largely localized to helices F and G, and provides an 'opening' of the protein to protons on the cytoplasmic side of the membrane.     

水杨醛甘氨酸Schiff碱的从头算构象研究

运用从头算HF方法对水杨醛甘氨酸Schiff碱的构象进行了理论研究.结果表明,C=N键与苯环的共轭以及分子内弱相互作用是分子能量降低的2个重要结构因素.在各存在形态的所有最稳定构象中c=N键均与苯环共轭,均至少存在1条H14的弱氢键.酸式结构最稳定构象羧基上羟基氢H22背离大共轭平面;酸根形态的稳定构象羧基与苯环共面形成平面分子,且存在2条氢键;钾盐酚式最稳定构象羧基螯合成键,而醌式最稳定构象为平面分子,羧基单齿成键,且存在H14的2条弱氢键.由于具有很大的能量优势,水杨醛甘氨酸Schiff碱在水中的主要存在形态为酸式,该结论与其水溶液的实验研究结果一致.     

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.     

环三磷腈对羟基苯甲醛芳胺衍生物的合成及分子识别功能

以环三磷腈为起始原料,在2,2-二羟基联苯的保护下合成了化合物（1）和（2）,并与对羟基苯甲醛芳氨希夫碱进一步反应,经分离纯化后得到四种环三磷腈衍生物。通过红外光谱、核磁共振波谱对化合物进行了结构表征,证实为目标产物。利用紫外光谱研究了四种衍生物对信号分子激活剂磷酸吡哆醛(PLP)的识别功能和剂-效关系。结果表明,希夫碱基枝化衍生物（6）和（8）具有较好的识别能力。     

Translocation step size and mechanism of the RecBC DNA helicase

DNA helicases are ubiquitous enzymes that unwind double-stranded DNA. They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice--DNA--by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3'-->5' direction. The translocating enzyme will traverse, or 'step' across, single-stranded DNA gaps in defined steps that are 23 (+/-2) nucleotides in length. This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins.     

希夫碱钼(Ⅵ)配合物的合成表征与生物活性研究

用一步法制备了N-氧化吡啶-2-甲醛缩氨基酸系列希夫碱及其钼（Ⅵ）配合物,并用元素分析、IR、差热、热重分析等进行了表征,结果表明,钼（Ⅵ）通过与希夫碱的O（N-氧化吡啶）、N（希夫碱）、O（羧基）原子配位形成稳定的配合物。生物活性试验表明：希夫碱对植物发芽有抑制作用,配合物则有促进作用,亦证明钼（Ⅵ）为活性中心。     

香兰素对甲苯胺希夫碱硝酸稀土配合物的合成

制备了香兰素(3-甲氧基-4-羟基苯甲醛)对甲苯胺希夫(Schiff)碱硝酸稀土配合物。该配合物由一个中心稀土离子,二个单齿配位希夫碱,二个双齿配位硝酸根和一个外界硝酸根组成,中心稀土离子的配位数是6,其一般式为[LnL_2(NO_3)_2]NO_3(Ln=La, Ce, Pr, Nd, Sm, Eu; L=希夫碱配体)。     

Cloning of a gene that is rearranged in patients with choroideraemia

Choroideraemia (tapetochoroidal dystrophy, TCD), a common form of X-linked blindness, is characterized by progressive dystrophy of the choroid, retinal pigment epithelium and retina. Previous studies have assigned the TCD gene to a small segment of the Xq21 band. By making use of reverse genetics strategies we have isolated eight overlapping complementary DNA clones from the same chromosomal region. The corresponding gene is expressed in retina, choroid and retinal pigment epithelium. The cDNAs encompass an open reading frame of 948 base pairs that is structurally altered in eight TCD patients with deletions, and in a female patient with a balanced translocation involving Xq21. These findings provide strong evidence that we have cloned the gene underlying choroideraemia. Elucidation of its function should provide new insights into the molecular mechanisms responsible for this disorder and other hereditary retinopathies.     

Pumping of protons from the mitochondrial matrix by cytochrome oxidase

The stoichiometry and mechanism of redox-linked proton translocation by the mitochondrial respiratory chain is a major issue of debate in membrane bioenergetics. The function of cytochrome oxidase is a focal point of disagreement. In 1977 it was suggested that the terminal component of the respiratory chain, cytochrome oxidase, functions as a redox-linked proton pump. That and subsequent studies were based mainly on measurements of proton ejection from mitochondria or from vesicles reconstituted with isolated cytochrome oxidase, or on measurements of translocation of electrical charge equivalents across mitochondrial and vesicle membranes. This proton-translocating function of cytochrome oxidase is confirmed here by a quantitative determination of proton uptake from the inside (matrix) of intact mitochondria.     

四齿席夫碱类光学探针用于检测铀和含磷酸基团生物分子的研究进展

席夫碱具有易制备、检测金属离子灵敏度高和选择性好等特点,长期以来受到人们的广泛关注。近年来四齿席夫碱类光学探针在医学、催化、分析化学、超分子化学、功能材料及配位化学等领域得到广泛应用。本文结合实时热点,阐述了四齿席夫碱类光学探针利用其特有的NN或NO型空腔与铀酰离子配位,实现对实际样品中铀酰离子的定性分析和定量检测,并成功应用于含磷酸基团生物分子的识别与检测。最后展望并探讨了四齿席夫碱类光学探针的研究发展方向。     

Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 A resolution.

The X-ray crystal structure of the molecular complex of penicillin G with a deacylation-defective mutant of the RTEM-1 beta-lactamase from Escherichia coli shows how these antibiotics are recognized and destroyed. Penicillin G is covalently bound to Ser 70 0 gamma as an acyl-enzyme intermediate. The deduced catalytic mechanism uses Ser 70 0 gamma as the attacking nucleophile during acylation. Lys 73 N zeta acts as a general base in abstracting a proton from Ser 70 and transferring it to the thiazolidine ring nitrogen atom via Ser 130 0 gamma. Deacylation is accomplished by nucleophilic attack on the penicilloyl carbonyl carbon by a water molecule assisted by the general base, Glu 166.     

N—2—羟在苄叉对氯苯胺及其铜配合物的合成

合成了新化合物席夫碱N－2－羟在苄叉对氯苯胺及对甲基苯氧基二硫代甲酸铜。用红外光谱对所合成样品进行了表征。将席夫碱分别与丙二酸铜,邻苯二甲酸铜,对甲基苯氧基二硫代甲酸铜及与二乙基二硫代氨甲酸铜4种铜盐配位后溶于乙醇中生长晶体。通过对所得晶体进行元素分析后发现,该席夫碱与4种铜 盐的配位方式不完全相同,由于邻苯二甲酸铜的螯合性能较强,只有一个席夫碱分子与其配位,而其他3种铜盐与席夫碱反应时,有2个席夫碱与铜离子配位,且席夫碱上的羟基取代了原理铜盐上的阴离子。     

带有巯基的席夫碱在金电极表面的自组装成膜

利用自组装技术,将带有巯基的席夫碱成功地自组装到Au电极表面,并利用循环伏安和交流阻抗电化学技术,对自组装膜的性质进行了表征。结果显示,单独用席夫碱成膜,由于分子结构的不规则性,形成的膜较疏松;采用二次成膜的方法,制备了由席夫碱和十八硫醇构成致密的复合膜,并通过实验和计算证明复合膜中的席夫碱呈线形方式排列,并且未被十八硫醇所取代。通过自组装膜,用电化学方法深入地研究了席夫碱中的电子传输机理、席夫碱与金属离子的络合机理、席夫碱在生物体中的活性等问题。     

Banach空间中的广义m—增生映象

1　ＰｒｅｌｉｍｉｎａｒｉｅｓＴｈｒｏｕｇｈｏｕｔｔｈｉｓｐａｐｅｒ ,ｌｅｔＥｂｅａｒｅａｌＢａｎａｃｈｓｐａｃｅｗｉｔｈｄｕａｌｓｐａｃｅＥ ,2 Ｅ ｄｅｎｏｔｅｔｈｅｆａｍｉｌｙｏｆａｌｌｔｈｅｎｏｎｅｍｐｔｙｓｕｂｓｅｔｓｏｆＥ ,ａｎｄ〈· ,·〉ｄｅｎｏｔｅｔｈｅｄｕａｌｐａｉｒｂｅｔｗｅｅｎＥａｎｄＥ .ＬｅｔＡ :Ｅ → 2 Ｅｂｅａｍｕｌｔｉｖａｌｕｅｄｍａｐｐｉｎｇ .ＤｅｆｉｎｅｔｈｅｎｏｒｍａｌｉｚｅｄｄｕａｌｉｔｙｍａｐｐｉｎｇＪ :Ｅ→ 2 Ｅ ｂｙＪ(ｘ) ={ ｆ∈Ｅ :〈ｘ ,ｆ〉 =‖ｆ‖ ·‖ｘ…     

三氮唑,噻二唑和四唑席夫碱的植物激素活性

首次对三氮唑，噻二唑和四唑Schiff碱的生物活性进行了系统报道，生物活性测试结果发现，这些Schiff碱大多数具有明显的植物激素活性，其中三氮唑的Schiff碱具有优良的细胞分裂素活性，四唑的Schiff碱具有很好的生长素活性与生根性能，而噻二唑的激素活性则稍差，生测试验结果还证明，氯代苯甲醛，噻吩甲醛和吲哚甲醛生成的Schiff碱生物活性更好。     

双水杨醛缩苯二胺锌荧光量子效率的研究

研究了4种双水杨醛缩苯二胺锌配合物的荧光性能,测定了它们的荧光量子效率.结果表明,不同取代基对双水杨醛缩苯二胺锌的荧光效率有不同影响:配合物中供电子取代基使双西佛碱金属配合物的荧光效率增强,吸电子取代基使双西佛碱金属配合物的荧光效率降低.为设计合成具备高荧光量子效率的发光材料提供了依据.     

金刚烷胺缩3,5-二溴水杨醛Schiff碱的合成、表征及抗菌活性研究

首先用NaOH的乙醇溶液处理金刚烷胺盐酸盐,得到金刚烷胺.金刚烷胺再与3,5-二溴水杨醛脱水生成金刚烷胺缩3,5-二溴水杨醛Schiff碱(IUPAC命名:N-3,5-二溴亚水杨醛基金刚烷胺,以下简称Schiff碱).Schiff碱用元素分析、红外光谱、核磁共振氢谱等手段进行了表征.测试了四个不同浓度的Schiff碱对金黄色葡萄球菌及大肠杆菌抗菌作用.试验结果表明,当Schiff碱溶液浓度为1.0×10-1mo1.L-1时对金黄色葡萄球菌表现出较强的抑菌作用,当浓度为1.0×10-4mo1.L-1时,表现出较弱的抑菌作用;四个不同浓度Schiff碱对大肠杆菌没有抑菌作用.