表面带负电荷氨基酸残基的突变对细胞色素bs与细胞色素c结合和识别的影响

利用核磁共振方法研究表面带不同负电荷氨基酸残基突变后的细胞色素b5与细胞色素c的结合与识别，结果表明，静电作用在细胞色素b5与细胞色素c的结合过程中有着重要的贡献，而且这些静电贡献在一定程度上具有 加性，E48的贡献略大于E44。同时还证明Brownian dyanmics simulations优化出的Glu48-Lys13,Clu56-Lys87,Asp60-Lys86和heme 6-propionate-Tml72（细胞色素b5的残基排在前面）的结合方式在溶液中的确存在，细胞色素b5突变体9E48，E56／A，D60／A）及[Cr(oxalate)3]^3－对细胞色素c的表面竞争实验表明，细胞色素c表面结合区SiteⅢ仍然同细胞色素b5突变体9E48，E56／A，D60／A）有结合作用，只是结合强度上相对于野生细胞色素b5同细胞色素c的结合有所降低，这表明除上述的Browni-an dynamics simulations模型外，尚有其它如Salemme模型等的结合方式，这也揭示出细胞色素b5和细胞色素c之间的结合是比较动态的。     

表面带负电荷氨基酸残基的突变对细胞色素b5与细胞色素c结合和识别的影响

利用核磁共振方法研究表面带不同负电荷氨基酸残基突变后的细胞色素b₅与细胞色素c的结合与识别.结果表明,静电作用在细胞色素b₅与细胞色素c的结合过程中有着重要的贡献,而且这些静电贡献在一定程度上具有累加性,E48的贡献略大于E44.同时还证明Browniandynamicssimulations优化出的Glu48-Lys13,Glu56-Lys₈₇,Asp60-Lys86和heme6-propionate-Tml₇₂(细胞色素b₅的残基排在前面)的结合方式在溶液中的确存在.细胞色素b₅突变体(E48,E56/A,D60/A)及[Cr(oxalate)₃]^3-对细胞色素c的表面结合竞争实验表明,细胞色素c表面结合区Site仍然同细胞色素b₅突变体(E48,E56/A,D60/A)有结合作用,只是结合强度上相对于野生细胞色素b₅同细胞色素c的结合有所降低.这表明除上述的Brownian dynamics simulations模型外,尚有其它如Salemme模型等的结合方式,这也揭示出细胞色素b₅和细胞色素c之间的结合是比较动态的.     

细胞色素b5-蛋白质相互作用研究

蛋白质-蛋白质相互作用在生命过程中发挥至关重要的作用,特别是血红素类蛋白。细胞色素b5(Cyt b5)是血红素蛋白的一个典型代表,在生物体内通过多种蛋白质-蛋白质相互作用来执行其生物功能。目前所揭示的与Cyt b5相关的蛋白质相互作用包括:细胞色素b5-细胞色素b5还原酶,细胞色素b5-细胞色素P450,细胞色素b5-细胞色素c,细胞色素b5-肌红蛋白或血红蛋白,细胞色素b5-融合蛋白(谷胱甘肽S-转移酶GST和绿色荧光蛋白GFP)和细胞色素b5-转运蛋白(蔗糖转运蛋白SUT1和山梨醇转运蛋白SOT6)等。同一蛋白能与众多不同蛋白相互作用的事实,使我们认识到某些特定蛋白的生物学重要性。另一方面,研究同一蛋白与不同蛋白质间的相互作用将会进一步加深我们对蛋白质结构与功能关系的理解,以及指导新颖蛋白的理性设计与最终应用。     

突变对嗜热古菌蛋白[P62A]Ssh10b主链构象影响的核磁共振研究

嗜热古菌蛋白Ssh10b突变体[P62A]Ssh10b具有良好的热稳定性. [P62A]Ssh10b的结构测定结果显示, 其α2螺旋上残基K48和D51之间形成了一对盐键, 而且, 突变D51将影响蛋白的热稳定性. 为了探索D51的突变对蛋白热稳定性的影响, 构建了突变体[D51N/P62A]Ssh10b的质粒, 并获得了高纯度的15N和13C双标记[D51N/P62A]Ssh10b. 通过对异核三共振NMR实验数据的解析, 完成了对[D51N/P62A]Ssh10b的主链共振近乎完全的指认. 比较突变以及未突变蛋白质的主链1HN和15N化学位移, 在[P62A]Ssh10b的结构基础上进行分析发现, D51N突变显著地影响了α2螺旋骨架构象, 并进一步影响到古菌Lβ2α2loop区域、β4 的N端区域、Lβ3β4 loop的C端区域以及β3与Lβ3β4 loop的交界区域. 结果表明, 由于D51N突变破坏了α2螺旋上K48和D51之间的盐键, 影响了[D51N/P62A]Ssh10b的α2螺旋构象, 并影响到蛋白的其它相关部位的局部构象, 说明 [P62A]Ssh10b 的高热稳定性可能与其溶液构象密切相关. 为进一步运用NMR研究[D51N/P62A]Ssh10b分子结构特性与耐热机制间的关系奠定了基础.     

基于3D-药效团的P糖蛋白和细胞色素P4503A4的协同作用机理研究

P糖蛋白(P-glycoprotein,Pgp)和细胞色素P4503A4(CYP3A4)是决定药物ADME性质的两个重要蛋白,目前还无法通过实验方法,从分子水平清晰阐明这两个蛋白采取怎样的互补作用机理来降低外来药物的生物利用度.通过3D-药效团模建方法,提取Pgp和CYP3A4的共同底物的特征阐明这两个蛋白可能的协同作用模式.所得的药效团有助于理解药物分子同这两个蛋白的作用模式,同时该模型可以指导新药设计和改造,从而提高药物的生物利用度.     

细胞色素b5突变体V45H NMR初步研究

通过分析在H2O和D2O中采集,DQF-COSY,TOCSY和NOESY等二维核磁共振波谱鉴定了细胞色素b5定点突变体V45H(残基Val^45突变为His^45)的大多数氨基酸残基的质子自旋系统,通过解析NOESY谱中的dNN(i,i+1),dαN(i,i+1),dαN(i,i+2),dαN(i,i+3),dαβ(i,i+3)和dβN(i,i+1)等NOE相关,完成了其序列特异性归属以及主链和侧链质子共振信号的全归属。突变体V45H的二级结构分析表明残基Val^45突变为His^45对分子的整体折叠影响不大。但是,与野生型细胞色素b5相比较,突变体V45H主链酰胺质子的化学位移指数提示突变使其血红素疏水腔的微环境受到扰动。以上实验结果为进一步测定V45H的溶液结构和分析残基Val^45在蛋白质中的作用提供了基础。     

4-羟基脯氨酸立体异构对α-芋螺毒素溶液构象的影响

在α-芋螺毒素及其它家族的芋螺毒素中, 脯氨酸的羟基化是非常普遍的后转录修饰方式. 在天然芋螺毒素中脯氨酸的羟基常采用反式构型, 且该残基对芋螺毒素的结构与生物活性产生了重要的影响, 而顺式构型的羟脯氨酸对α-芋螺毒素的折叠与生物活性的影响还鲜有研究. 本工作通过二维(2D)溶液核磁共振方法测定了经过化学修饰的三个含有反式或顺式羟脯氨酸的α-芋螺毒素的溶液结构, 它们是α4/7亚家族芋螺毒素肽[γ15E]Sr1B、[O7O'/γ15E]Sr1B和[O6O'/γ14E]Vc1A. 研究表明, 羟基顺反异构化学修饰对芋螺毒素的结构影响显著. 羟脯氨酸羟基的反式到顺式修饰导致α-芋螺毒素肽明显的溶液构象变化, 这些变化包括二级结构的改变、关键残基的侧链取向变化以及氢键性质的改变. [O7O'/γ15E]Sr1B与[γ15E]Sr1B相比, 典型的α-芋螺毒素ω弯曲结构发生形变. 而[O6O'/γ14E]Vc1A不同于Vc1A的是末端转角结构的缺失. 本工作加深了对α-芋螺毒素肽的化学修饰法的理解, 该方法是阐明α-芋螺毒素结构-生物活性关系的有用工具.     

Rps. viridis光合反应中心分子间相互作用对电子转移机理影响的理论研究

基于晶体结构并经QM/MM优化后的结构数据, 用量子化学密度泛函(DFT, B3LYP)方法, 在6-31G基组水平上, 对紫色光合细菌Rhodopseudomonas(Rps.)viridis 反应中心内, 色素分子与蛋白质环境中氨基酸残基以及水分子间的配位及氢键等相互作用对反应中心原初电子转移反应机理的影响进行了探讨. 结果表明: 组氨酸残基的轴向配位使色素分子的ELUMO显著升高, 这对电子转移能够进行极为重要; 而氢键作用使色素分子的E_LUMO有所降低, 有利于说明电子转移由原初电子给体P沿L分支进行. 文中结果支持电子转移反应为不经过辅助细菌叶绿素的一步过程. 只将色素分子周围的蛋白质环境作为具有一定介电常数的均匀介质来处理是远远不够的.     

细胞色素b5突变体P40V血红素微环境核磁共振研究

细胞色素b5是一个具有多种重要生理功能的低自旋金属蛋白, 作为呼吸链系统的主要电子传递体之一受到了广泛的重视. 为了研究细胞色素b5表面疏水斑区Pro⁴⁰在细胞色素b5结构与功能上的重要性, 本文利用定点突变技术把细胞色素b5中Pro⁴⁰突变为Val⁴⁰, 并采用1D与2D ¹H NMR的方法对其进行分析, 探讨了Pro⁴⁰突变为Val⁴⁰对细胞色素b5血红素微环境的影响.     

(E)-4-硫-5-(2-溴乙烯基)尿苷/脱氧尿苷的合成及细胞毒性评价

以尿苷(1a)或2'-脱氧尿苷(1b)类化合物为原料,经碘代、选择性氧化、Heck反应等步骤快捷、环保地合成(E)-5-(2-溴乙烯基)尿苷/(E)-5-(2-溴乙烯基)-2'-脱氧尿苷(5a/5b),再通过酯化、硫羰基化、氨解等步骤合成新化合物(E)-4-硫-5-(2-溴乙烯基)尿苷/(E)-4-硫-5-(2-溴乙烯基)-2'-脱氧尿苷(8a/8b),共得到5种新的硫代产物,其中包含2种新产物8a/8b,3种新中间体7a,7b,10.其结构通过~1H NMR,~(13)C NMR,IR,UV,HRMS,X-Ray等谱图手段进行了表征.采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-溴化四唑(MTT)实验方法对8a及其类似物进行了细胞毒性评价,发现8a/8b是潜在的抗肿瘤药物.     

Atomic Force Microscopy of Azobenzene-Derived Alkanethiolate Monolayers on Gold

Self-assembled monolayer of two azobenzene-derived alkanethiols with different terminal alkyl chain lengths (shortly as C₈C₃SH and C₁C₄SH) on gold were studied using atomic force microscopy. The C₈C₃SH SAMs showed a very regular packing structure with a lattice constant of a=0.57±0.04 nm, b=0.73±0.06 nm and θ=120±10°, mainly domianted by the terminated alkyl chains. On the other hand, the C₁C₄SH SAMs exhibited a number of different packing domains on the gold surface. The closest packing lattice was a=0.37±0.04 nm, b=0.47±0.02 nm, and θ=,107±6°, most probably representing the aggregation of azobenzene chromophoren. Another two typical lattices observed were a=0.65±0.03 nm, b=0.72±0.03 nm, θ=120±2°, and a=0.47±0.03 nm.b=0.47±0.03 nm.θ=100±10°, respectively. The packing structure of C₁C₄SH monolayers on gold is believed to be dominated by the azobenzene chromophore.     

在线二维液相色谱法同时测定婴幼儿和成人配方营养品中的维生素A、D3和E

建立了在线二维液相色谱同时快速测定婴幼儿配方乳品和成人强化乳品中维生素A、D₃和E含量的方法。首先,依据疏水减法模型,选择C8柱和极性嵌合的反相C18柱分别作为一维和二维分离柱,构成正交分离体系,并均以甲醇、乙腈和水作为流动相,检测波长设为263 nm(维生素D₃)、296 nm(维生素E)和325 nm(维生素A)。采用双三元液相色谱的左泵作为一维分析泵,完成维生素A、E的定量和维生素D₃的净化;根据维生素D₃在一维色谱柱上的保留时间,确定切割时间窗口,并以500 μL定量环收集含有维生素D₃的馏分,由双三元液相色谱的右泵将馏分带到二维色谱柱中,以维生素D₂作为内标物,采用内标法完成维生素D₃的定量分析,整个过程在密闭系统中自动化完成。在上述优化条件下测定了婴幼儿和成人奶粉、奶酪及酸奶等强化乳品中3种维生素的含量。经过1.25 kg/L KOH溶液的热皂化和石油醚的萃取,样品萃取液直接进样分析,得到维生素D₃的加标回收率为75.50%~85.00%,并通过配对t检验法与标准方法测定结果进行比较分析,结果差异无统计学意义,表明本方法可同时快速、准确测定婴幼儿及其他配方营养品中维生素A、D₃、E的含量,提高了样品分析效率。     

Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study

Abstract— The dynamics of the flavin bound to the flavocytochrome b₂ from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13-fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 10⁹ M ^-1 s^-1 when the flavin is bound to the enzyme and 8.3 × 10⁹ Mv s^-1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b₂ is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.     

EFFECTS OF UV IRRADIATION ON A LIVING SKIN EQUIVALENT

Abstract— The Living Skin Equivalent (LSE™) is an organotypic coculture composed of human dermal fibroblasts interspersed in a collagen-containing matrix and overlaid with human keratinocytes forming a stratified epidermis. The LSE has a dry, air-exposed epidermal surface suitable for the application of oils, creams and emulsions. These features suggested its feasibility as an in vitro skin model for studying the protective effects of sunscreens. Using the thiazolyl blue (MTT) conversion assay as a measure of mitochondrial function, the extent of cytotoxicity induced by various doses of UV-R (280–400 nm) or UV-A (320–400 nm) was evaluated in the LSE. The doses of UV radiation that caused 50% reductions in MTT conversion (UV-R₅₀ or UV-A₅₀) in different lots of LSE were 0.053 ± 0.021 J/cm² (n = 29) and 11.6 ± 4.9 J/cm² (n = 17) for UV-R and UV-A, respectively. The protective effects of an 8% homosylate standard and of five UV-A sunscreens, topically applied to the LSE, were determined and compared with their reported protection factors in human skin. Morphological changes and the release of proinflammatory mediators (interleukin-1-α, tumor necrosis factor-α and prostaglandin E2) implicated in UV-induced erythema were also demonstrated in the LSE exposed to UV-A or UV-B. The data suggest that the LSE can be used for studying the effects of U V radiation on skin and may have utility for assessing the efficacy of certain sunscreens against UV-B and UV-A.     

SINGLET-OXYGEN GENERATION AT GAS-LIQUID INTERFACES: A SIGNIFICANT ARTIFACT IN THE MEASUREMENT OF SINGLET-OXYGEN YIELDS FROM OZONE-BIOMOLECULE REACTIONS

Abstract— Several ozone-biomolecule reactions have previously been shown to generate singlet oxygen in high yields. For some of these orone-biomolecule reactions, we now show that the apparent singlet-oxygen yields determined from measurements of 1270 nm chemiluminescence were artifactually elevated by production of gas-phase singlet oxygen. The gas-phase singlet oxygen results from the reaction of gas-phase ozone with biomolecules near the surface of the solution. Through the use of a flow system that excludes air from the reaction chamber, accurate singlet-oxygen yields can be obtained. The revised singlet-oxygen yields (mol ¹O₂ per mol O₃) for the reactions of ozone with cysteine, reduced glutathione, NADH, NADPH, human albumin, methionine, uric acid and oxidized glutathione are 0.23 ± 0.02, 0.26 ± 0.02, 0.48 ± 0.04, 0.41 ± 0.01, 0.53 ± 0.06, 1.11 ± 0.04, 0.73 ± 0.05 and 0.75 ± 0.01, respectively. These revised singlet-oxygen yields are still substantial.     

灵芝菌丝体多糖的化学组成和溶液性质

用磷酸盐缓冲液在80℃时从灵芝菌丝体中提取出多糖-蛋白质缀合物LM-A.成分分析表明,GLM-A主要由葡萄糖组成,蛋白质含量为13%.GLM-A在1∶1水稀释的饱和镉乙二胺溶液中30℃时的特性粘数[η]和均方根旋转半径〈S²〉^1/2的分子量依赖关系分别为[η]=5.1×10^-2M_w^0.06(cm³·g^-1)和〈S²〉^1/2=3.9×10^-2M_w^0.50(nm).按照Yamakawa-Fujii-Yoshizaki蠕虫状圆筒模型的粘度理论和Bohdanecky表达式,求得GLM-A的分子参数为:单位围长摩尔质量M_L=(530±10)nm^-1,持久长度q=(2.8±0.2)nm.链直径d=0.75nm.实验结果表明,GLM-A在该溶液中为无规线团构象.     

A CALORIMETRIC DETERMINATION OF THE QUANTUM YIELD FOR THE JONIZATION OF MALACHITE GREEN CYANIDE BY ULTRAVIOLET RADIATION

Abstract The quantum yield for the conversion of malachite green cyanide (MGL) to the oxidized form MG⁺ has been measured over the wavelength range from 225 to 289 nm using a total absorption aluminium calorimeter to measure the flux of photons. The number of molecules of MGL converted was determined from the increase in absorbance of the solution at 622 nm. MG⁺ was found to have a maximum extinction coefficient of 10.63 × 10⁴ at 622 nm. The quantum yield for the conversion of MGL to MG⁺ is constant over the wavelength range with a value of 0.91 ± 0.01. The use of MGL as an actinometer for photochemical studies is described.     

EFFECT OF MID-UV RADIATION ON DNA-Cu2+ COMPLEX: ABSORPTION AND CIRCULAR DICHROISM STUDY

Abstract— When DNA is complexed with Cu²⁺ ions it becomes sensitized to mid-UV light. Irradiation of the complex at 310 ± 10 nm produces marked changes in absorption and circular dichroism spectra in addition to those in melting profiles of irradiated samples. These variations show conformational changes in DNA structure due to a photoreduction of some copper sites in the DNA-Cu²⁺ complex. In addition to this effect an irreversible damage in DNA structure could be invoked to explain the observed behaviour. These facts can be related to the presence in DNA-Cu²⁺ complex of a small absorption band extending up to mid-UV range while the absorption of DNA is negligible in this spectral range.     

DETECTION OF THE EXCITED SINGLET STATE OF A DEPROTONATED, REDUCED FLAVIN

Abstract— The excited singlet state of a deprotonated, reduced flavin [1, 5-dihydro- N (3)-carboxymethyllumiflavin] in aqueous solution at pH 8 has been detected by laser flash photolysis. The broad absorption band maximized at ∼ 490 nm (ε= 9.9 × 10³ M ^-1 cm^-1). The lifetime of the transient was found to be 100 ± 15 ps. The lifetime was not affected by the presence of pyrimidine dimers, which would be monomerized under these conditions. A longer-lived transient, tentatively identified as the solvated electron, was also detected. The neutral reduced flavin did not give a detectable transient.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.