电子顺磁共振测量蛋白质分子内选定位点之间距离的方法及应用

蛋白质分子的结构决定了其特性和功能,准确测量蛋白质分子中特殊位点之间的距离对其结构解析至关重要。该文在简要介绍电子顺磁共振(electron paramagnetic resonance,EPR)方法测量蛋白质分子内未偶自旋之间距离基本原理的基础上,重点综述了近年来EPR结合定点自旋标记(site-directed spin label,SDSL)技术在研究蛋白质结构与功能方面的应用情况,归纳了EPR-SDSL方法测量距离的特点和存在问题,并提出了改进用连续波EPR技术测量距离的准确度的思路和实现方法。     

分子运动性预测麻栎种子离体胚轴适宜贮藏条件初探

应用电子顺磁共振波谱仪和自旋标记技术,以硝基氧探针为标记物,检测了室温下麻栎种子离体胚轴脱水过程中分子运动性的变化。发现含水量0.7gH2O/gDW至0.64gH2O/gDW范围是细胞质粘度的转折区域,低于这个含水量区域,细胞质粘度骤然上升,推测这个区域是室温下保存离体胚轴的适宜含水量下限。通过变温电子顺磁共振波谱测定,找到离体胚轴含水量在0.43gH2O/gDW至1.02gH2O/gDW范围内,分子运动性的临界温度和玻璃态相变温度所在区间。根据分子运动性随温度变化的规律,预测含水量为0.69gH2O/gDW的麻栎种子离体胚轴适宜贮藏温度约为-50℃。     

脱水导致种子和花粉细胞膜变化的生物热力学研究进展

膜系统是引起脱水敏感细胞的损伤和死亡的原初位点之一,该文综述了近年来对以下各问题以及它们之间的关系的研究进展:(1)膜相变;(2)膜系统和生物大分子受到的压力;(3)玻璃态的形成及其部位;(4)造成玻璃态形成的溶质分子的大小对膜相变的影响;(5)两性物质在细胞质水相和膜脂脂相间的再分配.旨在为如何有效地监控种子和花粉寿命和最大程度地减少种子和花粉细胞的衰老损伤,预测种质保存的最佳贮藏条件和种质寿命,以便及时更换保存样本等方面的研究提供新的思路.     

Photo-induced electron transfer between hypocrellins and nano-sized semiconductor CdS——EPR study on the kinetics of photosensitized reduction in HA-CdS and HB-CdS systems

Both HA-CdS and HB-CdS (Hys-CdS, Hys represents HA, HB) complex systems were established according to the dynamics of heterogeneous electron-transfer process μ = Es/s+-ECB <0. In these systems, the electron transferring from 1Hys* to conduction band ofCdS is feasible. Determined from the fluorescence quenching, the apparent association constants (Kapp) between Hypocrellin A(HA), Hypocrellin B (HB) and CdS sol. were about 940 (mol/L)-1, 934 (mol/L)-1, respectively. Fluorescence lifetime measurements gave the rate constant for the electron transfer process from 1HA*, 1HB* into conduction band of CdS semiconductor as 5.16×109 s-1, 5.10×109 s-1, respectively. TEMPO (2,2,6,6-tetramethy-1-piperdinyloxy), a stable nitroxide radical, was used in the kinetic study of the reduction reaction taking place on the surface of a CdS colloidal semiconductor, kinetics equation of the reaction was determined with the electron paramagnetic resonance (EPR) method, and the reaction order of TEMPO is zero. When Hys were add     

种子大小对小泡巨鼠贮藏行为的影响

以小泡巨鼠为研究对象,在围栏条件下,测定小泡巨鼠对大小两种栓皮栎种子的贮藏行为差异,以检验最优贮藏空间模型的两个预测:(1)大种子(高价值食物)的贮藏率大于小种子(低价值食物);(2)大种子的贮藏距离远于小种子.结果表明:大种子的搬运比例(39%)多于小种子(21.5%),多数的小种子(53%)被取食,大种子的贮藏比率(16%)远大于小种子(7%),上述结果支持模型的预测(1).但是,不同大小种子在贮藏距离上没有显著差异,因而预测(2)没有被支持.实验结果表明种子价值对鼠类的取食和贮藏行为有十分重要的影响,但围栏空间可能影响对模型预测(2)的检验.     

苝醌类光敏剂HA, HB与纳米半导体CdS间的光生电子传递——HA-CdS, HB-CdS复合体光敏还原动力学的EPR研究

根据非均相体系电子传递动力m = Es*/s+ - ECB (m＜0 的原理, 构建出相匹配的HA-CdS和HB-CdS复合体(以下简称Hys-CdS, Hys代表HA, HB). 在该体系中, 1Hys*向CdS导带传递电子是热力学允许的. 通过荧光淬灭实验, 测出它们之间的表观结合常数(Kapp)分别为940 (mol/L)-1(HA-CdS), 934 (mol/L) -1(HB-CdS), 表明Hys与CdS间存在较强烈的吸附效应. 荧光寿命测定得1Hys*向CdS导带传递电子的速率常数Ket分别为5.16×109 s-1(HA-CdS), 5.10×109 s-1(HB-CdS). 以一种稳定的氮氧自由基TEMPO(2, 2, 6, 6-tetramethyl-1-piperdinyloxy)作为电子受体, 当它接受一个电子和一个质子, 其EPR(electron paramagnetic resonance)信号便会消失, 据此, 应用消自旋的EPR技术, 定量研究了苝醌类光敏剂Hys与纳米半导体CdS间光生电子传递动力学, 确定了受Hys光敏化作用的CdS纳米半导体表面光还原速率方程和反应动力学速率常数, 结果发现, 本体系中TEMPO接受电子的反应级数均为0, 而不同于均相体系中的反应级数, 特别是在相同可见光照射条件下, 通过比较单独的CdS与Hys-CdS复合体的光还原速率常数还发现, 后者的光还原效率比前者均大, 约为350倍, 表明Hys对CdS纳米半导体具有显著的光敏化作用. 这提示在太阳能应用中, Hys是一种很有效的光敏化剂.     

基于质子MRSI的肌细胞内脂测量技术的原理、现状及展望

胰岛素抵抗作为诱发糖尿病的主要原因可能早在糖尿病发病十多年前就已经出现.及早发现胰岛素抵抗对于糖尿病的早期预防、诊断、治疗有着至关重要的意义.研究发现,人类小腿肌肉细胞内脂质(intramyocellular lipid, IMCL)的含量与胰岛素抵抗和代谢障碍有着密切而又复杂的关联.因此,准确测量IMCL对于Ⅱ型糖尿病乃至多种精神疾病具有重要的研究意义.然而,目前测量IMCL的"金标准"是创伤性的活检技术,不适于大规模的基础研究和长期的临床应用.基于质子磁共振波谱成像(1H magnetic resonance spectrosocpic imaging,1H MRSI)的IMCL测量技术提供了一种无辐射、无创伤、高精度的检测方法,有望成为明晰IMCL含量与胰岛素抵抗,以及胰岛素抵抗与Ⅱ型糖尿病关系的重要技术并在临床上应用.本文基于1H MRSI的IMCL含量测量技术,阐述并总结了其发展历程,从小腿内部生理结构特征、肌纤维方向、脂肪分布、精确量化IMCL含量等方面指出目前该技术面临的挑战和最新的解决思路.本文特别探讨了多模态磁共振成像(magnetic resonance imaging, MRI)技术以及面向参数估计的磁共振指纹技术MRF(magnetic resonance fingerprinting)等新技术与质子磁共振波谱成像技术的结合,希望为IMCL高精度测量提供更加多样化、多维度、多尺度的可能和途径.     

胡杨种子萌发过程中几种相关酶的活性变化

在室温条件下,胡杨种子保留冠毛贮藏可适当延长其生命力;室温贮藏带冠毛的胡杨种子,前20 d萌发率降低幅度不大.随着种子贮藏时间的延长,过氧化物酶(POD)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性均先上升后下降,峰值出现时间与种子室温贮藏20 d相对应;脱氢酶活性先上升后下降再回升,峰值出现时间也与种子室温贮藏20 d相对应;酸性磷酸酯酶(ACP)活性与种子萌发率同步下降.室温贮藏40 d的带冠毛胡杨种子仍有22.3%的萌发率.     

不同无性系蔗茅抽穗开花特性的观察和花粉贮藏条件的研究

来源于较低海拔地区的3个蔗茅无性系的抽穗期较短,来源于较高海拔(大于1500m)地区的16个蔗茅无性系的抽穗期较长,且抽穗期与原产地海拔呈显著负相关(F=-0.827**).蔗茅的这一特性在集中种植后仍能相对稳定地表现出来.蔗茅花粉量较多,花粉发育良好.在自然条件下,蔗茅新鲜花粉仅能存活1 h左右;而经低温(10℃)硅胶干燥2 h后贮藏于-80℃条件下,其生活力保持可超过360d.蔗茅贮藏花粉生活力的下降比率与花粉含水量呈显著正相关(r=0.933**);含水量降至10%以下的花粉在低温中贮藏时才能保持较高生活力.     

陶塔维人的年代

法国东比利牛斯陶塔维(Tautavel)的阿拉戈洞(Cave of I'Arago)发现的人化石的年代,以前用热释光法(thermoluminescence)测得的年代为大约距今20万年。1983年横山(Yokoyama)用电子(回辶)旋共振法(electron spin resonance,ESR)测得的年代为大约距今50万年,比用热释光法所得的数据大一倍以上。最近英国的斯金纳(A.F.Skinner)和德贝纳姆(N.C.Debenham)对此数     

Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen

Although the occurrence of intracellular glasses in seeds and pollen has been established, physical properties such as rotational correlation times and viscosity have not been studied extensively. Using electron paramagnetic resonance spectroscopy, we examined changes in the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl during melting of intracellular glasses in axes of pea (Pisum sativum L.) seeds and cattail (Typha latifolia L.) pollen. The rotational correlation time of the spin probe in intracellular glasses of both organisms was approximately 10⁻³ s. Using the distance between the outer extrema of the electron paramagnetic resonance spectrum (2A_zz) as a measure of molecular mobility, we found a sharp increase in mobility at a definite temperature during heating. This temperature increased with decreasing water content of the samples. Differential scanning calorimetry data on these samples indicated that this sharp increase corresponded to melting of the glassy matrix. Molecular mobility was found to be inversely correlated with storage stability. With decreasing water content, the molecular mobility reached a minimum, and increased again at very low water content. Minimum mobility and maximum storage stability occurred at a similar water content. This correlation suggests that storage stability might be at least partially controlled by molecular mobility. At low temperatures, when storage longevity cannot be determined on a realistic time scale, 2A_zz measurements can provide an estimate of the optimum storage conditions.     

Effect of Long-Term Storage on Water Status and Physicochemical Properties of Nutritionally Enhanced Tortillas

The effect of functional ingredients (carrot juice, whole soy flour, and whole kamut flour) and storage (180 days) on physicochemical properties (texture and amylopectin recrystallization) and water status (moisture content, water activity, ice melting peak thermal properties, and proton nuclear magnetic resonance (¹H NMR) mobility) of tortillas has been studied. Different formulations significantly changed the parameters studied during storage resulting in larger changes than in the standard formulation (STD) that, therefore, may be considered the most stable product. The properties of whole kamut tortillas were very similar to those of standard sample while the formulation that contained carrot juice lead to an increased system rigidity observable both at macroscopic (textural properties), macromolecular (significantly reduced), and molecular (¹H FID) levels. A decrease of moisture content, water activity, endothermic transition ~0 °C, and an increase of ¹H NMR mobility (¹H T₂ pop A and C) were observed in soy-containing products [(soy enriched (SOY) and carrot, soy, and kamut (CSK)]. SOY and CSK had very low water activity, presented the highest ¹H NMR molecular mobility and underwent the most marked changes during storage suggesting that water activity cannot be taken as a sole indicator of food stability as very important modifications occurred in tortillas at molecular level.     

Structural elucidation and properties of 8alpha-(N1-histidyl)riboflavin: the flavin component of thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase.

In addition to 8alpha-(N3-histidyl)riboflavin, 8alpha-(N1-histidyl)riboflavin is also formed during the reaction of Nalpha-blocked histidine with 8alpha-bromotetraacetylriboflavin in a yield of 20-25% of the total histidylflavin fraction. The properties of 8alpha-(N1-histidyl)riboflavin are inditical with those of the histidylflavin isolated from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase but differ from those of 8alpha-(N3-histidyl)riboflavin. These properties include pKa of fluorescence quenching, electrophoretic mobility at pH 5.0, stability to storage, and reduction by NaBH4. Proof for 8alpha substitution is shown by the electron paramagnetic resonance and electron-nuclear double resonance spectra of the cationic semiquinone form, as well as by the proton magnetic resonance spectrum of the oxidized form. The site of histidine substitution by the 8alpha-methylene of the flavin moiety was shown by methylation of the imidazole ring with methyl iodide, cleavage of the methylhistidine-flavin bond by acid hydrolysis at 150 degrees C, and identification of the methylhistidine isomer by electrophoresis. 3-Methylhistidine is the product from the N1-histidylflavin isomer, while 1-methylhistidine is produced from the N3 isomer. The flavin product from reductive Zn cleavage of either isomer has been identified as riboflavin. The compound obtained on acid treatment of 8alpha-(N3-histidyl)riboflavin (previously thought to be the N1 isomer) differs from the parent compound only in the ribityl side chain, since chemical degradation studies show 1-methylhistidine as a product and a flavin product which differs from riboflavin only in mobility in thin-layer chromatography, but not in absorption, fluorescence, and electron paramagnetic resonance spectral properties. Proof that acid modification involves only the ribityl chain has come from the observations that alkaline irradiation of this flavin yields lumiflavin, that the proton magnetic resonance spectrum of the compound differs from that of riboflavin in the region of the ribityl proton resonance, and that its periodate titer is lower than that of authentic riboflavin. The identity of 8alpha-(N1-histidyl)riboflavin with the histidylflavin from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase shows that both isomeric forms of 8alpha-histidylflavin occur in nature.     

Astaxanthin binding and structural stability of the apple snail carotenoprotein ovorubin

Ovorubin (OR) is the major perivitellin of the eggs of Pomacea canaliculata. The astaxanthin (ASX) binding and structural stability of OR were investigated by fluorescence spectroscopy and circular dichroism (CD). The apo-OR (without astaxanthin) shows a single, high affinity binding site for ASX (K(D)=0.5 microM). The quenching of tryptophan fluorescence by ASX indicates that about 22% are near the carotenoid-binding site in a non-polar environment, as indicated by tryptophan resonance energy transfer to the ligand. Secondary structure (alpha+beta) was virtually not affected by cofactor removal. Holo-OR shows unusually high thermal stability. The removal of ASX does not affect the thermal or chemical stability of the quaternary structure. In conclusion, although subtle changes were observed, ASX is not essential for OR stability, unlike most invertebrate carotenoproteins. This supports the idea that OR plays an important physiological role in the storage, transport and protection of carotenoids during snail embryogenesis.     

Shoulder function: the perfect compromise between mobility and stability

Shoulder function is a compromise between mobility and stability. Its large mobility is based on the structure of the glenohumeral joint and simultaneous motion of all segments of the shoulder girdle. This requires fine-tuned muscle coordination. Given the joint's mobility, stability is mainly based on active muscle control with only a minor role for the glenohumeral capsule, labrum and ligaments. In this review factors influencing stability and mobility and their consequences for strength are discussed, with special attention to the effects of morphology, muscle function and sensory information.     

Intracellular glasses and seed survival in the dry state

So-called orthodox seeds can resist complete desiccation and survive the dry state for extended periods of time. During drying, the cellular viscosity increases dramatically and in the dry state, the cytoplasm transforms into a glassy state. The formation of intracellular glasses is indispensable to survive the dry state. Indeed, the storage stability of seeds is related to the packing density and molecular mobility of the intracellular glass, suggesting that the physico-chemical properties of intracellular glasses provide stability for long-term survival. Whereas seeds contain large amounts of soluble non-reducing sugars, which are known to be good glass formers, detailed in vivo measurements using techniques such as FTIR and EPR spectroscopy reveal that these intracellular glasses have properties that are quite different from those of simple sugar glasses. Intracellular glasses exhibit slow molecular mobility and a high molecular packing, resembling glasses made of mixtures of sugars with proteins, which potentially interact with additional cytoplasmic components such as salts, organic acids and amino acids. Above the glass transition temperature, the cytoplasm of biological systems still exhibits a low molecular mobility and a high stability, which serves as an ecological advantage, keeping the seeds stable under adverse conditions of temperature or water content that bring the tissues out of the glassy state.     

Comparison of the action of streptomycin and neomycin on the structure of the bacterial ribosome

The influence of streptomycin and neomycin upon the conformation of the ribosome has been investigated using spin-labeled and fluorescent analogs of the sulfhydryl reagent, N-ethylmaleimide. Changes in the electron paramagnetic resonance spectra or in the polarization of fluorescence of labeled ribosomes reveal that streptomycin alters the mobility of labels bound to the sulfhydryl group of protein S18 while neomycin affects the mobility of labels bound to the sulfhydryl groups of proteins S1, S21 and/or L10. It is also observed that both streptomycin and neomycin interfere with changes in the mobility of labels induced by storage under inactivating conditions. From these results, it is concluded that: 1. streptomycin and neomycin distort the conformation of the ribosome at different sites, streptomycin disturbing preferentially the area around the sulfhydryl group of protein S18 while neomycin affects the environment of the sulfhydryl groups of proteins S1, S21 and/or L10; 2. streptomycin and neomycin interefere with the ability of the ribosome to undergo conformational changes.     

Effect of the chemical surface structure of functionalized polystyrene on the kinetic properties of immobilized yeast pyruvate decarboxylase]

The influence of the loading density of different functional groups and the length of suitable spacer structures on the kinetic properties of yeast pyruvate decarboxylase are investigated on identical polystyrene matrices. At constant concentrations of the fixed protein both the specific activity and the storage stability of the immobilized enzymes increase with increasing concentrations of the protein binding (C = O)-groups. pH-Optimum and K'M-value prove to be functions of the NH3+-content of the supports. Using four spacer resins with an equal content of spacer groups it could be shown that the optimum time of coupling as well as the maximum catalytical activity, storage stability and thermostability depend on the length of the spacer structures. On the other hand, the mobility of an ESR-marker fixed via the same spacers to the resin is not affected by the different spacer structures.     

Protein inactivation in amorphous sucrose and trehalose matrices: effects of phase separation and crystallization

Trehalose is the most effective carbohydrate in preserving the structure and function of biological systems during dehydration and subsequent storage. We have studied the kinetics of protein inactivation in amorphous glucose/sucrose (1:10, w/w) and glucose/trehalose (1:10, w/w) systems, and examined the relationship between protein preservation, phase separation and crystallization during dry storage. The glucose/trehalose system preserved glucose-6-phosphate dehydrogenase better than did the glucose/sucrose system with the same glass transition temperature (T_g). The Williams-Landel-Ferry kinetic analysis indicated that the superiority of the glucose/trehalose system over the glucose/sucrose system was possibly associated with a low free volume and a low free volume expansion at temperatures above the T_g. Phase separation and crystallization during storage were studied using differential scanning calorimetry, and three separate domains were identified in stored samples (i.e., sugar crystals, glucose-rich and disaccharide-rich amorphous domains). Phase separation and crystallization were significantly retarded in the glucose/trehalose system. Our data suggest that the superior stability of the trehalose system is associated with several properties of the trehalose glass, including low free volume, restricted molecular mobility and the ability to resist phase separation and crystallization during storage.     

Is there a role for oligosaccharides in seed longevity? An assessment of intracellular glass stability

We examined whether oligosaccharides extend seed longevity by increasing the intracellular glass stability. For that purpose, we used a spin probe technique to measure the molecular mobility and glass transition temperature of the cytoplasm of impatiens (Impatiens walleriana) and bell pepper (Capsicum annuum) seeds that were osmo-primed to change oligosaccharide content and longevity. Using saturation transfer electron paramagnetic resonance spectroscopy, we found that the rotational correlation time of the polar spin probe 3-carboxy-proxyl in the cytoplasm decreased, together with longevity, as a function of increasing seed water content, suggesting that longevity may indeed be regulated by cytoplasmic mobility. Osmo-priming of the seeds resulted in considerable decreases in longevity and oligosaccharide content, while the sucrose content increased. No difference in the glass transition temperature was found between control and primed impatiens seeds at the same temperature and water content. Similarly, there was no difference in the rotational motion of the spin probe in the cytoplasm between control and primed impatiens and bell pepper seeds. We therefore conclude that oligosaccharides in seeds do not affect the stability of the intracellular glassy state, and that the reduced longevity after priming is not the result of increased molecular mobility in the cytoplasm.