枯草芽孢杆菌双精氨酸转运系统TatA_y蛋白的溶液结构（英文）

存在于细菌和植物叶绿体中的双精氨酸(Tat)蛋白质转运系统能将底物蛋白以折叠的状态进行跨膜转运.该系统中的单次跨膜膜蛋白Tat A通过自身寡聚形成转运底物蛋白的通道.该文应用液体核磁共振方法解析了枯草芽孢杆菌Tat Ay蛋白在十二烷基胆碱磷酸胶束中的结构,它是由一个跨膜螺旋(TMH)和一个两亲性螺旋(APH)构成的L型结构.通过与已经报道的枯草芽孢杆菌Tat Ad蛋白的结构比较,该文能够鉴定出参与维持L型构象的重要氨基酸残基,并指出了Tat A蛋白家族中若干较为保守的结构特征.在此基础上,该文讨论了保守残基在Tat A通道形成过程中可能发挥的作用.     

两种微生物源谷氨酰胺转氨酶与底物特异性识别的全原子动力学模拟研究

微生物源谷氨酰胺转氨酶(Transglutaminase, TGase)能够催化蛋白间氨酰基转移促进蛋白交联,已经广泛应用于食品、轻工等多种领域.然而目前微生物源TGase与底物间基于长程作用力的分子特异性识别机制仍不清楚,理性改造困难.本研究以源自高茂源链霉菌(MTG)和源自枯草芽孢杆菌(BTG)的两种TGase为研究对象,使用分子对接技术构建酶与底物的结合复合体并使用分子动力学模拟解析两种TGase与底物识别的分子机制.结果显示两种TGase在底物识别过程高度类似,均由催化腔外周loop区域氨基酸残基通过范德华及静电作用固定底物整体,而后由位于催化腔底部催化三联体内Asp(MTG)或Glu(BTG)通过羧基侧链稳定反应发生局部空间构象并拉近底物Gln内γ-酰胺基与核心催化残基Cys间距,以便后续反应过程中TGase-底物中间体的生成.上述结果阐明了TGase与底物活性基团的分子识别模式,提出了两种TGase内作用于底物识别的氨基酸残基,为以后TGase的理性改造研究提供了理论基础.     

神经电信号和运动肌细胞之间的桥梁——烟碱型乙酰胆碱受体通道(NAChR)

 烟碱型乙酰胆碱受体(NAChR)是通过打开或关闭一个跨膜生成孔的门来控制神经和肌肉细胞间的电信号。这个通道是由两部分组成的:直接面向通道的内环(innerring)是由5个α螺旋互相缠绕形成的,它们成放射状地排列,这样就为离子形成了一个逐渐变窄的通道。这个通道的外环(outerring)是由15个α螺旋互相缠绕形成的,它们的作用是将内环和外部的油脂层隔离开来。通道的“门”是一个位于细胞膜中间的一个压缩的疏水性区域(constrictinghydrophobicgirdle),它是靠内环相邻的螺旋之间的微弱作用力而形成。     

强碱胁迫枯草芽孢杆菌芽孢致死的拉曼光谱研究

应用单细胞拉曼分析技术结合PCA方法,以枯草芽孢杆菌芽孢为对象,以强碱为诱导剂实时记录芽孢生理变化过程,探究芽孢在强碱胁迫下的致死机制。实验表明,枯草芽孢杆菌芽孢和其萌发后芽孢在一定范围均有一定的耐受强碱能力,但萌发后芽孢的耐受力明显下降,主要原因是释放了维持其抗性及稳定性的特有Ca2+-DPA的保护;芽胞受强碱胁迫也会有少量Ca2+-DPA释放过程的行为特征,但通过分析所记录的光谱数据结果表明,其释放行为不同于由L-丙氨酸引起的芽孢的正常Ca2+-DPA释放过程。通过PCA比较分析强碱胁迫芽孢和萌发后芽孢Ca2+-DPA释放过程,主要是强碱破坏芽孢膜结构,膜损伤后强碱比较容易进到芽孢内部从而破坏蛋白质的主链结构链及核酸;芽孢膜通道也可能受到强碱的伤害,造成微量Ca2+-DPA的释放。     

二价金属离子与YycFN相互作用的NMR研究

YycGF最早发现于枯草芽孢杆菌,是与细胞存活密切相关的双组分信号转导系统存在于少量低鸟嘌呤和胞嘧啶(G+C)含量的革兰氏阳性菌中,包括金黄色葡萄球菌和肺炎链球菌等人类病原菌,在外界环境刺激下,胞膜上的组氨酸激酶YycG通过自身组氨酸磷酸化活化,将磷酸根转移至反应调节蛋白YycF的N端调节区(YycF_N)使之磷酸化,调控下游基因表达,实现特定细胞应答反应.二价金属离子在双组分信号转导系统反应调节蛋白的磷酸化过程中起着非常关键的作用,但它们与YycF_N相互作用的机制尚不清楚.该文利用液体核磁共振(NMR)方法研究了Ca~(2+)、Mg~(2+)两种离子与YycF_N的相互作用,对详细的相互作用界面进行了分析,并计算了Ca~(2+)、Mg~(2+)与YycF_N的解离常数(K_d).发现金属离子的关键作用位点是Asp9、Asp16和Asp53等残基,蛋白的整体构象也发生了一定变化,为阐明二价金属离子在反应调节蛋白信号转导过程中的作用机制提供了重要线索.     

人类唾液蛋白N 端片段的构型

人类唾液蛋白Statherin 是含有43 残基的酸性磷酸化蛋白．此蛋白对磷酸钙具有较高的吸附性,已知其N 端15 残基的片段(SN-15)会吸附于磷灰石的表面．在该工作中,作者以液体核磁共振波谱学研究SN-15 的分子结构．圆二色光谱显示SN-15 在磷酸盐缓冲溶液中具α 螺旋构型．根据高分辨核磁共振氢谱(COSY,TOCSY 及NOESY)作者得到相关的NOE 及J 耦合数据,氢氘交换实验也进一步显示SN-15 具α 螺旋构型．     

阵列式等离子体射流处理芽孢的实验研究北大核心CSCD

研制了一套单极性微秒脉冲阵列式等离子体射流系统,该系统可在大气压下激发产生等离子体射流,实现大面积的灭菌处理。该系统可产生峰值电压20 kV、频率15 kHz的高压脉冲,激发产生的射流均匀稳定,覆盖面积达37.7 cm2,射流长度达6 cm,射流功率为40.05 W,处理5 min可使射流覆盖范围内的枯草芽孢杆菌的芽孢基本全部失去活性。考察了不同参数对灭菌效率的影响,结果表明,灭菌率与工作电压、脉冲频率、处理时间呈正相关,在氦气氛围下有较好的灭菌效果。SEM显示等离子体射流能够对枯草芽孢杆菌的芽孢外壳结构造成损坏,导致芽孢无法正常代谢,最终死亡。     

蜡样芽孢杆菌的二阶导数红外光谱研究

采用傅里叶变换红外光谱非破坏性地分析了蜡样芽孢杆菌,在获得完整全细胞组分的红外光谱信息基础上,进行了二阶导数光谱转换。结果表明,根据二阶导数光谱特征吸收峰可区分蜡样芽孢杆菌细胞的荚膜、芽孢、贮能物质等特殊结果物质。以1 654 cm-1附近的α-螺旋结构的蛋白酰胺带吸收峰与在1 601和1 403 cm-1附近显示的强羧基伸缩振动吸收峰可探测到细胞荚膜结构;根据1 617,1 372和1 569 cm-1附近的吡啶二羧酸(DPA)的吸收峰可认定细胞内芽孢的存在;此外,可在二阶导数红外谱图中同时找到多聚β-羟基丁酸(PHB)细胞粒、荚膜、芽孢的吸收峰。由二阶导数红外光谱可分辨重叠光谱来探测细胞多种结构物质,为从分子生物学与细胞生物学角度,为研究蜡样芽孢杆菌提供参考信息。     

快中子辐照对枯草芽孢杆菌DNA损伤研究

采用脉冲场凝胶电泳技术检测并定量分析了CFBR-Ⅱ快中子脉冲堆产生的快中子在不同剂量和剂量率条件下, 对枯草芽孢杆菌黑色变种(ATCC 9372)DNA双链断裂的诱导. 通过DNA释放百分比PR值、DNA断裂水平L值、断裂DNA平均分子量和DNA片段分布等指标的分析, 结果表明：在不同的辐射条件下, DNA片段均明显分布于两个区域, 表明枯草芽孢杆菌黑色变种DNA分子上可能存在对中子辐射较为敏感的位点; 并且随着中子辐射剂量和剂量率的变化, DNA~释放百分比PR值、DNA断裂水平L值和各片段区双链断裂的含量也会发生一定规律性的变化.     

杆状病毒蛋白融合肽LdF在溶液中的NMR结构研究

LD130是舞毒蛾核多角体病毒（Lymantria dispar multiple nucleopolyhedrovirus, LdMNPV）的膜融合蛋白（F蛋白）,其F₁亚基N端疏水的保守区为介导膜融合过程的活性肽段,即融合肽区域. 利用核磁共振的方法,确定了该融合肽在酸性条件下类膜环境中的溶液结构. 结果表明融合肽LdF具有典型的α螺旋结构,整个肽段于类膜环境中呈现两亲性,即螺旋沿轴向可分为亲水侧面和亲脂侧面. 该结构有利于对病毒囊膜与细胞膜融合过程的深入理解与研究.     

Protein structural codes and nucleation sites for protein folding

One of the long-standing controversial arguments in protein folding is Levinthal's paradox. We have recently proposed a new nucleation hypothesis and shown that the nucleation residues are the most conserved sequences in protein. To avoid the complicated effect of tertiary interactions, we limit our search for structural codes to the nucleation residues. Starting with the hypotheses of secondary structure nucleation and conservation of residues important for folding, we have analysed 762 folds classified as unique by SCOP. Segments of 17 residues around the top 20% conserved amino acids are analysed, resulting in approximately 100 clusters each for the main secondary structure classes of helix, sheet and coil. Helical clusters have the longest correlation range, coils the shortest (four residues). Strong specific sequence-structure correlation is observed for coil but not for helix and sheet, suggesting a mapping relationship between the sequence and the structure for coil. We propose that the central sequences in these clusters form `structural codes', a useful basis set for identifying nucleation sites, protein fragments stable in isolation, and secondary structural patterns in proteins (particularly turns and loops).     

A naturally occurring omega current in a Kv3 family potassium channel from a platyhelminth

Background  

Voltage-gated ion channels are membrane proteins containing a selective pore that allows permeable ions to transit the membrane in response to a change in the transmembrane voltage. The typical selectivity filter in potassium channels is formed by a tetrameric arrangement of the carbonyl groups of the conserved amino-acid sequence Gly-Tyr-Gly. This canonical pore is opened or closed by conformational changes that originate in the voltage sensor (S4), a transmembrane helix with a series of positively charged amino acids. This sensor moves through a gating pore formed by elements of the S1, S2 and S3 helices, across the plane of the membrane, without allowing ions to pass through the membrane at that site. Recently, synthetic mutagenesis studies in the Drosophila melanogaster Shaker channel and analysis of human disease-causing mutations in sodium channels have identified amino acid residues that are integral parts of the gating-pore; when these residues are mutated the proteins allow a non-specific cation current, known as the omega current, to pass through the gating-pore with relatively low selectivity.     

超声处理黑豆蛋白结构变化的拉曼光谱分析

探究了拉曼光谱应用于黑豆蛋白结构变化研究的可行性,研究了黑豆蛋白溶液在低频超声处理在不同超声强度、不同处理时间下的结构变化,并进行了热力学特性分析。低、中超声处理强度下TD的降低表明蛋白质分子的内部疏水作用被破坏,使黑豆蛋白不稳定的聚集体解聚为小分子可溶性聚集物,而在高超声处理强度下TD的增高表明聚集体重聚。拉曼光谱分析表明超声处理下除了E样品(300 W, 24 min)所有黑豆蛋白均发生了α-螺旋结构含量降低和β-折叠结构含量增高。聚集体的聚合/解聚导致黑豆蛋白二级结构的重组,尤其是β-折叠。超声处理使拉曼光谱在760 cm-1的色氨酸归属谱线强度降低表明超声处理使黑豆蛋白发生了部分的解折叠。超声处理下酪氨酸归属谱线强度变化不显著,表明超声处理并未显著改变黑豆蛋白酪氨酸的微环境。1 450 cm-1拉曼归属谱带随着超声处理强度和时间的增加而增大,但随着功率及处理时间的进一步增大此值有所降低。在超声处理下聚集体的形成使二硫键的g-g-t构型转变为t-g-t构型。尽管黑豆蛋白聚集体重组的机理仍有待研究,但拉曼光谱是一种研究超声处理黑豆蛋白结构变化的可行方法,也可为蛋白质结构研究提供一种新的研究思路。     

Elasticity of alpha-helical coiled coils

Predicting large scale conformations of protein structures is computationally demanding. Here we compute the conformation and elasticity of double-stranded coiled coils using a simple coarse-grained elastic model. By maximizing the contact between hydrophobic residues and minimizing the elastic energy, we show that the minimum energy structure of a coiled coil is a supercoiled double helix of alpha helices. For realistic binding energies, the elastic energy of the alpha helices requires binding every 7th residue, which leads to a pitch and helix angle for the structure that is consistent with experimental measurements. Analysis of the model equations shows how the pitch varies with the helical repeat of the hydrophobic residues and with the ratio of the twisting modulus to the bending modulus and provides an estimate of the persistence length of around 150 nm, in agreement with previous experimental estimates.     

A solid-state NMR index of helical membrane protein structure and topology

The secondary structure and topology of membrane proteins can be described by inspection of two-dimensional (1)H-(15)N dipolar coupling/(15)N chemical shift polarization inversion spin exchange at the magic angle spectra obtained from uniformly (15)N-labeled samples in oriented bilayers. The characteristic wheel-like patterns of resonances observed in these spectra reflect helical wheel projections of residues in both transmembrane and in-plane helices and hence provide direct indices of the secondary structure and topology of membrane proteins in phospholipid bilayers. We refer to these patterns as PISA (polarity index slant angle) wheels. The transmembrane helix of the M2 peptide corresponding to the pore-lining segment of the acetylcholine receptor and the membrane surface helix of the antibiotic peptide magainin are used as examples.     

Ethanol-induced conformational fluctuations of NMDA receptors

Alcohol addiction ranks among the leading global causes of preventable death and disabilities in human population. Understanding the sites of ethanol action that mediate its acute and chronic neural and behavioural effects is critical to develop appropriate treatment options for this disorder. The N-methyl-d-asparate (NMDA) receptors are ligand-gated heterotetrameric ion channels, which are known to directly interact with alcohol in a concentration-dependent manner. Yet, the exact molecular mechanisms and conformational dynamics of this interaction are not well understood. Here, we conducted a series of molecular dynamics simulations of the interaction of moderate ethanol concentrations with rat's wild-type GluN1–GluN2B NMDA Receptor under physiological conditions. The simulations suggest that glutamate or glycine alone induce an intermediate conformational state and point towards the transmembrane domain (TMD) as the site of action of ethanol molecules. Ethanol interacts by double hydrogen bonds with Trp635 and Phe638 at the transmembrane M3 helix of GluN2B. Alcohol not only reduces the pore radius of the ion channel within the TMD but also decreases accessibility of glutamate and glycine to the ligand-binding sites by altering the structure of the ligand-binding domain and significantly widening the receptor in that area.     

Solid-state 17O NMR spectroscopy of a phospholemman transmembrane domain protein: implications for the limits of detecting dilute 17O sites in biomaterials

The 17O-'diluted' glycine-14 sites in a phospholemman (PLM) transmembrane domain protein are characterized by solid-state 17O NMR spectroscopy. The PLM transmembrane domain is an alpha-helical tetramer unit of four 28-residue peptides and is rigidly embedded in a bilayer where each alpha-helix has an average tilt of 7.3 degrees against the membrane normal. The PLM sample investigated here consists of a high lipid/peptide molar ratio (25:1) with one glycine residue in each helix enriched to <40% (17)O; thus, this is a very dilute 17O-sample and is the most dilute 17O-membrane protein to date to be characterized by solid-state 17O NMR spectroscopy. Based on the spectral analysis of 17O magic angle spinning (MAS) at 14.1 and 18.8T, the PLM transmembrane domain protein consists of multiple crystallographic gly14 sites, suggesting that the tetramer protein is an asymmetric unit with either C2- or C1-rotational symmetry along the bilayer normal.     

绿荧光蛋白的双光子激发的荧光特性研究

利用双光子激发方式研究了重组绿荧光蛋白（recombinant green fluorescent protein,简称rGFP)的光转换特性,研究结果表明rGFP具有较强的双光子激发荧光,双光子激发的荧光偏振光谱表明rGFP在辐照前质子态和去质态之间存在着有效的能量转移过程,rGFP辐照后导致生色团构象的变化,部分阻断了rGFP内源的氨基酸与生色团之间的能量转移过程,导致rGFP的双光子激发的荧光强度下降,观察到rGFP的三光子激发的荧光特性,这种三光子激发的荧光主要来源于rGFP内源的氨基酸（色氨酸,酪氨酸等）的吸收。研究结果对在实际使用定量的光显微镜时具有一定的指导意义。     

The impact of adsorption of bovine pancreatic trypsin inhibitor on CTAB‐protected gold nanoparticle arrays: a Raman spectroscopic comparison with solution denaturation

The influence of an array of cetyltrimethylammonium bromide (CTAB)‐protected gold nanoparticles on the structure of a model protein, bovine pancreatic trypsin inhibitor (BPTI) at pH 7.4, was studied by Raman spectroscopy. The structural consequences of array adsorption were compared with the effects of deposition of the protein directly onto a roughened gold substrate and with thermal and reductive treatment of BPTI in solution. Both thermal and reductive denaturation in solution result in loss of α‐helix structure, with an increase in random conformations of the protein in the case of reductive denaturation and β‐sheet conformation and random coil on thermal denaturation. For reductive denaturation in particular, extensive loss of secondary structure is evident. Deposition of the protein onto the array resulted in increased β‐sheet conformation similar to that observed on thermal treatment of the protein. However, unlike denaturation, which for both thermal and reductive process resulted in changes in the disulfide stretching wavenumber, this remains largely unchanged on application of the protein to the array. Furthermore, deposition of the protein onto bare gold results in significant heterogeneity in the S S stretching signal with appearance of ggt and nonequilibrium geometry of the CCSSCC dihedral angles. Thermal denaturation results in a red shift of the SS mode, whereas dithiothreitol (DTT) treatment, as expected, induces significant loss of the S S stretching signal, although a signal at 517 cm⁻¹ remains suggesting that the unreduced disulfide has changed to the ggt geometry. In addition, an SH mode is observed at 2570 cm⁻¹ in solution. The response of BPTI to thermal and DTT treatment while on the array is very different to its solution behavior, and suggests that adhesion to the array increases the stability of the protein. Copyright © 2009 John Wiley & Sons, Ltd.     

拉曼光谱法研究不同环境温度下脉冲电场对胰岛素二级结构影响及理论模型分析

近年来,弱电磁辐射生物效应已受到人们的普遍关注.前期研究发现脉冲电场与环境温度对胰岛素分子的构象产生协同效应.因此,文章首先应用拉曼光谱法研究了不同环境温度下脉冲电场对胰岛素分子二级结构的影响,得到胰岛素分子α螺旋结构含量的变化;并运用蛋白质螺旋-线团结构转变模型对实验结果进行定量分析,得到的理论模型可以较好地刻画不同环境温度下,脉冲电场对胰岛素分子α螺旋结构含量的影响.脉冲电场的作用及热力学环境的改变,使蛋白质螺旋结构向无规线团结构发生转变,可以解释脉冲电场作用下,胰岛素分子α螺旋结构含量随环境温度升高而下降的原因.研究结果为进一步研究弱电磁辐射对生物大分子二级结构的非热效应机理提供了一定的实验依据及理论参考.