Probing the Conformational Stability of Two Different Copper Proteins: A Dynamic Fluorescence Study on Azurin and Ascorbate Oxidase

Tryptophan fluorescence is extremely useful to monitor structural conformational transitions in proteins. Denaturant-induced unfolding of azurin and ascorbate oxidase has been studied by dynamic fluorescence measurements in the frequency domain and the results have been interpreted in terms of continuous distribution of lifetimes. The data add new information on the unfolding mechanism that was previously analyzed by steady-state emission spectroscopy. In particular, the existence of multiple, parallel unfolding pathways may be envisaged and correlated, in both cases, to the two protein structures. The effect of metal depletion has been also characterized by fluorescence lifetime measurements. In the case of azurin, a monomeric protein, the data demonstrate that copper removal yields a totally different unfolding pathways with respect to the holo protein, indicating that metal ion plays a fundamental structural role in the wild type, native protein. In the case of ascorbate oxidase a dimer of 140 kDa, only minor effects have been detected by copper removal. However, the analysis of the fluorescence decay in presence of different amounts of guanidinium hydrochloride gives new important insights on the unfolding intermediates. In particular the data support the hypothesis of a partial exposure of an outer layer of dimer at intermediate denaturant concentration. This ability of dynamic fluorescence to pinpoint the presence of structural micro-heterogeneity in the unfolding pathways of proteins demonstrates the greater power of this technique compared to the most commonly used steady-state measurements.     

酵母蛋白质网络的动力学性质

蛋白质-蛋白质、蛋白质-DNA相互作用网络决定了细胞中各种关键功能的执行．基于芽殖酵母(budding yeast saccharomyces cerevisiae)的蛋白质-蛋白质相互作用网络数据和相关的实验文献，我们建立了调控细胞周期和生命周期(cell cycle and life cycle)的蛋白质网络，并利用离散模型研究了该网络的动力学性质，研究表明：细胞周期网络的动力学性质具有很强的稳定性，约94％的蛋白质初态将演化到对应于生物学G1基态的稳定态，使其成为惟一的全局吸引点；同时，绝大多数的初态的演化路径都通过由G1激发态到G1基态的细胞周期演化路径，使细胞周期路径成为全局性的“吸引”路径。     

Nanosecond-time-resolved infrared spectroscopic study of fast relaxation kinetics of protein folding by means of laser-induced temperature-jump

Elucidating the initial kinetics of folding pathways is critical to the understanding of the protein folding mechanism. Transient infrared spectroscopy has proved a powerful tool to probe the folding kinetics. Herein we report the construction of a nanosecond laser-induced temperature-jump （T-jump） technique coupled to a nanosecond timeresolved transient mid-infrared （mid-IR） spectrometer system capable of investigating the protein folding kinetics with a temporal resolution of 50 ns after deconvolution of the instrumental response function. The mid-IR source is a liquid N2 cooled CO laser covering a spectral range of 5.0μm （2000 cm^-1）-6.5μm （1540 cm^-1）. The heating pulse was generated by a high pressure H2 Raman shifter at wavelength of 1.9μm. The maximum temperature-jump could reach as high as 26±1℃. The fast folding/unfolding dynamics of cytochrome C was investigated by the constructed system, providing an example.     

蛋白质INSM1中的锌指结构域ZF (4-5)的表达、纯化与表征

胰岛素瘤相关蛋白1（INSM1）是一类转录调节蛋白,通过其C-端的锌指结构域（氨基酸250-510）来识别序列特异性的DNA分子.INSM1的C-端包含有5个串联的锌指结构域,然而这些结构域的结构及其如何识别DNA的分子机制目前仍不清楚.通过重组构建的质粒pET-32m-INSM1（424-497）表达的蛋白质（氨基酸424-497）包含了最后两个锌指结构域4和5,简称为ZF（4-5）.该文详细研究了蛋白质ZF（4-5）的诱导表达条件,得到了较高产率的纯化蛋白.核磁共振（NMR）谱和圆二色谱（CD）揭示了Zn²⁺对稳定锌指蛋白结构的必要性,以及C2H2-Zn²⁺结合的组氨酸呈现为δ-异构方式.     

乳清分离蛋白-葡聚糖接枝物性质的荧光光谱法分析

乳清分离蛋白与葡聚糖的混合物在干热处理条件下,发生了以褐变为特征的美拉德反应。当葡聚糖分子量由67kD增至150kD时,游离氨基含量分别下降了35.77%和30.53%,糖链越长,其接入到蛋白质肽链的难度越大。采用荧光光谱对乳清分离蛋白-葡聚糖接枝物的性质进行分析。内源荧光光谱图显示,接枝产物在405nm的最大荧光强度显著提高,且350～500nm范围内的荧光强度顺序为:G67>G150,这说明接枝物中有Maillard反应体系所特有的荧光物质生成;由外援荧光光谱图得出,接枝产物在470nm的最大荧光强度均有明显降低,各溶液体系中荧光强度高低顺序依次为:WPI>G150>G67。疏水性指数的测定进一步说明两种不同分子量的葡聚糖接入到蛋白质肽链中,对乳清分离蛋白的疏水性均有一定的屏蔽作用。     

谷物种子中蛋白质含量测定的核反应分析法

分析了在高蛋白质谷物育种中对筛选方法的要求, 对谷物中蛋白质测定的方法进行了综述评价, 重点介绍了用于谷物蛋白质测定的核反应分析法的原理及特点. The requirements of the screening method for high protein content in grain breeding are analysed. Methods for the determination of protein content in grain seed are reviewed.The principle and the advantages of the nuclear reaction method for the measurement of protein content in grain seed are discussed in detail.     

An electron microscopic study of the archaeal feast/famine regulatory protein 5. Fourier filtration of images

Microcrystals of the feast/famine regulatory protein (FFRP) pot0434017 (FL11) were prepared by sonicating larger crystals. Using the microcrystals a cryo-electron micrograph was obtained, which showed a hexagonal packing of cylinder-like assemblies of FL11. This micrograph was processed by selecting, in the Fourier space, spots reflecting the crystal lattice, thereby removing the noise. The microcrystal was not totally free from distortion, and cylinders in local clusters adopted slightly different orientations. Thus, 25 hexagonal units closest to the ideal, each containing a cylinder at the center surrounded by six others, were manually selected. The averaged image was further processed to yield a perfect six-fold symmetry. These processed images, and some of the original images too, show bridges connecting cylinders, each corresponding to two pairs of N-domains, protruding from the two cylinders and contacting between them in the X-ray structure.     

TOF-SIMS structural characterization of self-assembly monolayer of cytochrome b5 onto gold substrate

Orientation and three-dimensional structure of immobilized proteins on bio-devices are very important to assure their high performance. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is able to analyze upper surface of one layer of molecules. Orientation of immobilized proteins can be evaluated based on determination of a partial structure, representing ensemble of amino acids, on the surface part. In this study, a monolayer of cytochrome b5 was reconstituted onto gold substrate and investigated by surface plasmon resonance (SPR). After freeze-drying, the resulted protein self-assembly was evaluated using TOF-SIMS with the bismuth cluster ion source, and then TOF-SIMS spectra were analyzed to select peaks specific to cytochrome b5 and identify their chemical formula and ensembles of amino acids. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the modified cytochrome b5 and three-dimensional structure of cytochrome b5 registered in the protein data bank. Finally, fragment-ion-generating parts of the immobilized-cytochrome b5 are determined based on the suggested residues and three-dimensional structure. These results suggest the actual structure and confirm the expected orientation of immobilized protein.     

不同龄期养殖鲈鱼的生化组成

对平均体重为1.22g,265g和1094g的一龄、二龄和三龄养殖鲈鱼肌肉中的水分，蛋白质、脂肪及氨基酸和脂肪酸含量进行了分析。结果表明，养殖鲈鱼肌肉中水分，蛋白质和脂肪的含量分别为76.39%-77.51%,17.68%-18.12%和3.56%-4.03%。鲈鱼肌肉的氨基酸总量占肌肉干重百分比在一龄，二龄和三龄养殖鲈鱼中分别为76.62%.75.11%和73.78%。平均为75.17%，必需氨基酸分别为40.10%,39.02%和36.69%。平均为38.60%，每克肌肉（湿重）含有脂肪酸总量在一龄、二龄和三龄养殖鲈鱼中分别为22.10mg,20.65mg和13.42mg。平均为18.72mg。其中PUFA总量分别为10.19mg,7.57mg和2.54mg，平均为6.77mg。     

甜菜糖蜜酒精废液生产菌体蛋白饲料初探

甜菜糖蜜酒精废液含有大量的有机物，pH值低，BOD、CDO值高，直接排和会造成环境的严重污染，又损失了一些有机物，在此废液中可直接接入酵母菌，使其大量繁殖，生产出的菌体粗蛋白含量达41．7％，CDO值降低了20％以上，减轻了环境污染。