W544F定点突变提高苏云金杆菌Cry1Ac蛋白的稳定性

W544是Cry1Ac蛋白上独特于其它Cry类蛋白的一个氨基酸, 它与F578和F604一起组成一个“螺旋桨状”的疏水簇, 通过疏水相互作用维持蛋白的三维结构稳定. 本研究通过定点突变将W544保守地替换为苯丙氨酸, SDS-PAGE分析结果表明其纯化的原毒素对紫外照射、胰蛋白酶处理和室温存贮的稳定性相对于野生Cry1Ac都有一定程度的提高; 经原子力显微镜观察, 发现W544F产生的晶体两个顶点间的垂直距离比野生型Cry1Ac约长0.6 μm, 且晶体表面不及野生型光滑; 此外, W544F与野生Cry1Ac的杀虫活性相似, 但经过紫外光照射9 h后, 其保留的杀虫活性比野生型高4倍以上. W544F突变较好地解决了Cry1Ac毒素蛋白田间应用不持久的问题, 具有重要的应用价值.     

基于细胞膜表面电势探讨Ca与毒性离子在植物根膜表面的相互作用

有毒重金属离子的自由离子活度已被广泛认为是重金属最直接的活性形态,可以用来预测重金属的生物效应,而事实上生物体表的重金属离子浓度由于细胞表面膜电势的静电效应而与本体溶液中重金属的自由离子活度存在显著差异.细胞膜表面电势随溶液中离子组成变化而发生变化,本体介质中钙离子能通过电荷屏蔽和离子键结合来降低膜表面电势的电负性,从而减少阳离子如Al3+、Cu2+和Ni2+在膜表面的活度,增加阴离子如SeO42-和H2AsO4-在膜表面的活度,这种电负性降低的程度能通过Gouy-Chapman-Stern模型来量化.基于植物根表重金属离子活度可以更好地预测其生物效应.细胞膜表面电势为研究离子之间交互作用机制及其与植物效应之间的关系提供了一个全新的角度.     

基于分子动力学模拟的TRPM8通道门控特性分析

TRPM8通道的温度感知等生理功能依赖于正常的门控, 但现有晶体结构中S6跨膜螺旋C末端形成的门控结构存在氨基酸缺失, 所以其门控特性未能揭晓. 本文基于已有的晶体结构和AlphaFold算法构建了 11个完整不同构象的TRPM8通道, 发现其S6跨膜螺旋C末端构成的门控存在回环和螺旋2种构象. 在回环构象中, 多个氨基酸参与形成阻碍离子通透的孔道区; 而在螺旋构象中, 仅有关键氨基酸V956发挥门控作用. 由于回环构象的柔性大于螺旋构象, 导致回环构象参与阻碍离子通透的关键氨基酸构象和数量变化多样. 二级结构预测与模建结果表明, S6跨膜螺旋C末端存在回环构象向螺旋构象的转变, 此过程中柔性的回环构象结构域向胞外侧上移, 关键氨基酸向孔道衬外扭转, 增强了与相邻跨膜螺旋S5的相互作用以及S5与TRP螺旋之间的相互作用, 进而形成刚性、 稳定且有序的螺旋构象. 这增加了TRPM8通道各结构域间的协同性, 使能量信息更高效地传递到门控结构域, 为TRPM8通道开启蓄势.     

三元配合物Zn(L-tyr)(Aa)分子内配体间疏水作用

应用pH电位滴定法研究了三元配合物Zn(L-tyr)(As)[L-tyr(酪氨酸根)，Aa=L-val(缬氨酸根)。L-php(苯丙氨酸根)]，L-trp(色氨酸根)]在水和x=0．20、0．40及0.60二氧六环-水混合溶剂中的稳定性[t=25℃。I=0．1mol／L NaClO4]。三元配合物相对稳定性用δK=ΔlogK[=logβZw^Zn(L-try)(Aa)-log KZn^Zn(L-try)-logKZn^Zn(Aa)]-ΔlogK(a)[=1/2ΔlogK(L-trp) ΔlogK(Aa) log2]表示。结果表明：与L-丙氨酸(L-ala)配合物Zn(L-tyr)(L-ala)相比，所有这些配合物均具有相对较高的δK值，表明配合物分子内存在着额外的稳定化作用。这种稳定性化作用可能主要归因于三元配合物分子内氨基酸侧链之间的疏水作用。并且这种作用随着氨基酸侧链结构及溶剂极性变化而变化。     

生物信息学分析人MEPE蛋白的结构与功能

通过生物信息学方法分析了人MEPE蛋白的理化性质、亚细胞结构定位、跨膜区、信号肽、空间结构、蛋白相互作用网络及MEPE分子的进化保守性等。分析表明，人MEPE蛋白等电点为8.62，理论上属于碱性亲水蛋白，有信号肽区域，但无跨膜区，预测为分泌型蛋白分子；主要二级结构元件是无规卷曲；MEPE在哺乳动物中高度保守，能与多种蛋白发生相互作用。     

转基因抗虫蛋白Cry1Ac与表面活性剂作用的芘探针光谱分析

以芘作为外源荧光探针,采用牛血清蛋白作为参照,考察了转基因抗虫蛋白Cry1Ac与4种表面活性剂相互作用的荧光光谱特征.结果表明,鼠李糖脂和Tween 80体系的芘荧光行为有相似的变化规律,可与蛋白质发生稳定的缔合过程.Cry1Ac蛋白的亲水性和分子极性较强,在表面活性剂浓度低时,可导致芘的I1/I3值较高.在阴离子、非离子表面活性剂胶团形成后,2种蛋白质的存在均不改变芘与表面活性剂的结合位点.十六烷基三甲基溴化铵对芘的荧光有一定猝灭作用,且在Cry1Ac蛋白介入下不能形成稳定的胶束.     

酶联免疫分析法探测Cry1Ab蛋白在不同介质中的构象变化

采用酶联免疫分析（ELISA）方法,根据构象变化后的蛋白与抗体结合能力下降从而导致ELISA测定值降低的原理,探测了转cry1Ab基因水稻表达的Cry1Ab蛋白在不同溶液介质中的热致构象变化行为,以及不同有机溶剂及溶液pH值对该蛋白构象变化的影响程度。实验表明,Cry1Ab蛋白在不同条件下的构象变化程度可以灵敏地通过ELISA方法检测。在不同的介质中,CrylAb蛋白的热致构象变化程度不同。在Na2SO4介质中,该蛋白具有较高的热稳定性;SDS的存在,可以促进该蛋白的构象变化。常温下,25％（V／V）的有机溶剂乙腈、异丙醇、甲醇、乙醇均能使该蛋白的构象发生转变,其中以乙腈最为显著。醇类溶剂对Cry1Ab蛋白的构象影响程度随疏水性增大而增大;溶液pH值也对该蛋白的构象变化产生影响。pH在8-10之间,该蛋白构象能保持稳定;酸或过碱性的溶液均能使蛋白构象偏离原始状态,从而引起ELISA测定值的降低。另外,腐殖酸能在一定时间内保持Cry1Ab蛋白构象的稳定性。     

顺磁共振和傅里叶变换红外光谱法研究高温对荒漠苔藓质膜结构影响

运用顺磁共振波谱和原位傅立叶变换显微红外光谱研究了高温对荒漠植物刺叶墙藓不同叶龄水合组织质膜透性和膜蛋白二级结构的影响。自旋标记法研究质膜透性结果表明,处理前野生叶质膜透性普遍高于室内培养获得的原丝体和次生叶,处理后次生叶膜透性变化最大,比处理前增加4倍,其次为原丝体。质膜透性随叶龄增加,变化幅度逐渐降低。红外光谱的二阶求导、傅立叶自解卷积及拟合分峰等结果显示,各叶龄间蛋白质二级结构含量差异较大,表明各龄组织蛋白成分不完全相同;高温处理后次生叶和原丝体α螺旋含量分别比对照增加40%和16%;其它叶龄组织二级结构含量变化范围小,表明热胁迫下老龄组织蛋白质二级结构稳定。膜透性和膜蛋白二级结构分析证明,膜透性和蛋白质稳定性呈正相关,指示蛋白质组成和含量不同是造成各龄组织不同耐热性的主要原因之一。     

Slc11a2第六跨膜区在十二烷基磷酰胆碱胶束中的结构

采用圆二色谱和核磁共振波谱研究了Slc11a2第六跨膜区在十二烷基磷酰胆碱(DPC)膜模拟环境中的结构和定位.结果表明,Slc11a2第六跨膜区在DPC胶束中N端和C端各形成一小段α螺旋,两段螺旋中间通过高度灵活的区域连接,整个肽插入到DPC胶束中.H267A突变使螺旋长度变长,H272A突变使螺旋长度略有变短,突变后结构的变化可能和蛋白功能缺失相关.     

蜂毒肽及其类似物的抗菌活性、溶血活性及与磷脂膜的作用

用CD谱测定了蜂毒肽及其类似物在Tris缓冲液、含2mol/LNaCl的Tris缓冲液和质量分数为10%的六氟异丙醇(HFIP)水溶液中的二级结构.结果表明,蜂毒肽及其类似物在Tris缓冲液中无确定的二级结构.在含2mol/LNaCl的Tris缓冲液中的α-螺旋结构的含量大大增加,表明其形成聚集体.未发现聚集与抗菌活性或溶血活性之间有相关性.在10%HFIP溶液(模拟生物膜环境)中的α-螺旋结构的含量大大增加,表明这些多肽具有内在形成α-螺旋结构的倾向.比较二级结构与抗菌和溶血活性发现,细菌细胞膜促进α-螺旋结构形成的环境比红血球细胞膜和HFIP水溶液强得多,而红血球细胞膜和HFIP水溶液的环境类似.     

Adsorption on montmorillonite prevents oligomerization of Bt Cry1Aa toxin

The adsorption of the insecticidal Cry1Aa protein from Bacillus thuringiensis (Bt-toxin) on a model clay surface was studied to understand the structural changes of the protein induced by the clay surface. We studied the adsorption of the monomeric and soluble oligomeric forms of the Cry1Aa toxin as a function of pH and ionic strength conditions on montmorillonite, which is an electronegative phyllosilicate. Cry1Aa secondary structure was determined from the amide I' FTIR absorption profiles. Accessibility to the solvent was determined by NH/ND exchange to characterize conformational flexibility of the different states of the Cry1Aa protein. The size distribution of Cry1Aa solutions was obtained by dynamic light scattering (DLS). From combined DLS and FTIR measurements, we conclude that montmorillonite traps the Cry1Aa toxin in its monomeric state, preventing the oligomerization of the protein. The oligomeric forms were adsorbed onto the clay without significant structural changes.     

Synergism of the Bacillus thuringiensis Cry1, Cry2, and Vip3 Proteins in Spodoptera frugiperda Control

The polyphagous caterpillar, Spodoptera frugiperda, has been controlled with either chemical insecticides or transgenic plants such as Bt maize that expresses the cry and/or vip genes of the Bacillus thuringiensis (Bt) bacterium. Despite the efficiency of Bt toxins in lepidopteran control, populations resistant to Bt plants have emerged in different locations around the world. Thus, understanding how combined proteins interact against pests can assist resistance control and management. This work demonstrated the toxicity of Cry1Ab, Cry1Ac, Cry1Ca, Cry1Ea, Cry2Aa, Cry2Ab, Vip3Aa, and Vip3Ca in single and combined assays against S. frugiperda neonatal larvae. All protein mixtures had synergistic action in the control of the larvae. The Vip3Aa + Cry1Ab mixture had the highest toxicity, sequentially followed by Vip3Aa + Cry2Ab, Cry1Ab + Cry2Ab + Vip3Aa, Cry1Ea + Cry1Ca, Cry1Ab + Cry2Ab, Vip3Ca + Cry1Ea, and Vip3Ca + Cry1Ca. Cry1Ab, Cry1Ac, Cry2Ab, and Vip3Aa bound to more than one site on the brush border membrane vesicles (BBMV) of S. frugiperda. The Cry1Ab and Cry1Ac proteins share binding site, while Cry1Ab does not share binding site with the Cry2Aa and Cry2Ab proteins. The Vip3Aa protein does not share receptors with the tested Cry1 and Cry2. The results suggest that combination these tested proteins may increase toxicity against S. frugiperda neonates.

     

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

Background  

To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays.     

Homology Modeling of Cry1Ac Toxin-binding Alkaline Phosphatase Receptor from Helicoverpa armigera and Its Functional Interpretation

Alkaline phosphatases (ALPs) attached to the midgut membrane with glycosyl phosphotidyl inositol (GPI) have been proposed as the putative Cry1Ac toxin receptor in Helicoverpa armigera. Activated toxins bind to ALP receptors on the brush border membrane vesicle (BBMV) of the midgut epithelium, which activates intracellular oncotic pathways and leads to cell death. However, with the long‐term use of Cry toxin, insects can develop a strong resistance to insecticidal delta‐endotoxins. Although the molecular mechanism of insect resistance has not been fully understood, insects develop resistance to biopesticides due to changes of toxins binding to midgut receptors. So, it is a good idea to investigate the molecular mechanism of insect resistance by analyzing ALP receptor from Helicoverpa armigera (Ha‐ALP). Based on crystal structure of shrimp alkaline phosphatase, the three‐dimensional structure of the Cry1Ac toxin‐binding Ha‐ALP receptor was obtained by homology modeling and the model was further evaluated using PROSA energy and ERRAT. The important role of binding of toxin to GalNAc on Ha‐ALP was discussed in the aspect of Cry1Ac toxicity. Specific recognition sites of the binding of oligosaccharides to Ha‐ALP were predicted. Post‐translational modification of ALP provides insights into the functional properties of ALP and leads to profound understanding of receptor and toxin interactions.     

In silico characterization of antifreeze proteins using computational tools and servers

In this paper, seventeen different fish Antifreeze Proteins (AFPs) retrieved from Swiss-Prot database are analysed and characterized using in silico tools. Primary structure analysis shows that most of the AFPs are hydrophobic in nature due to the high content of non-polar residues. The presence of 11 cysteines in the rainbow smelt fish and sea raven fish AFPs infer that these proteins may form disulphide (SS) bonds, which are regarded as a positive factor for stability. The aliphatic index computed by Ex-Pasy’s ProtParam infers that AFPs may be stable for a wide range of temperature. Secondary structure analysis shows that most of the fish AFPs have predominant α-helical structures and rest of the AFPs have mixed secondary structure. The very high coil structural content of rainbow smelt fish and sea raven fish AFPs are due to the rich content of more flexible glycine and hydrophobic proline amino acids. Proline has a special property of creating kinks in polypetide chains and disrupting ordered secondary structure. SOSUI server predicts one transmembrane region in winter flounder fish and atlantic cod and two transmembrane regions in yellowtail flounder fish AFP. The predicted transmembrane regions were visualized and analysed using helical wheel plots generated by EMBOSS pepwheel tool. The presence of disulphide (SS) bonds in the AFPs Q01758 and P05140 are predicted by CYS_REC tool and also identified from the three-dimensional structure using Rasmol tool. The disulphide bonds identified from the three-dimensional structure using the Rasmol tool might be correct as the evaluation parameters are within the acceptable limits for the modelled 3D structures.     

“Topohydrophobic positions” as key markers of globular protein folds

The positions of a given fold always occupied by strong hydrophobic amino acids (V, I, L, F, M, Y, W), which we call “topohydrophobic positions”, were detected and their properties demonstrated within 153 non-redundant families of homologous domains, through 3D structural alignments. Sets of divergent sequences possessing at least four to five members appear to be as informative as larger sets, provided that their mean pairwise sequence identity is low. Amino acids in topohydrophobic positions exhibit several interesting features: they are much more buried than their equivalents in non-topohydrophobic positions, their side chains are far less dispersed; and they often constitute a lattice of close contacts in the inner core of globular domains. In most cases, each regular secondary structure possesses one to three topohydrophobic positions, which cluster in the domain core. Moreover, using sensitive alignment processes such as hydrophobic cluster analysis (HCA), it is possible to identify topohydrophobic positions from only a small set of divergent sequences. Amino acids in topohydrophobic positions, which can be identified directly from sequences, constitute key markers of protein folds, define long-range structural constraints, which, together with secondary structure predictions, limit the number of possible conformations for a given fold. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 16 November 1998     

The functional domains of dopamine transporter for cocaine analog, CFT binding

Cocaine analogue, CFT (2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane) binding to dopamine transporter (DAT) in different species is quite heterogeneous. CFT is scarcely detected in bovine DAT whereas it is conspicuous in humans. To examine the structural basis for this functional discrepancy, we analyzed transporter chimeras of these two DATs. The CFT binding activities are avid in all of the chimeric DATs of which both of the 3rd and the 6-8th transmembrane domain (TM) are composed of human DAT sequences. On the contrary, CFT binding activities were scarcely detected if either or both of two regions are replaced with bovine sequences. These findings indicate that the CFT binding absolutely requires human DAT sequences, at least, in the regions encompassing the 3rd and 6-8th transmembrane domain (TM), and that these regions might contribute to form the 3-dimensional pocket for CFT binding.     

Nanobody-based electrochemical immunoassay for Bacillus thuringiensis Cry1Ab toxin by detecting the enzymatic formation of polyaniline

We describe an electrochemical immunoassay for the Cry1Ab toxin that is produced by Bacillus thuringiensis. It is making use of a nanobody (a heavy-chain only antibody) that was selected from an immune phage displayed library. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay where horseradish peroxidase (HRP) is used to produce polyaniline (PANI) from aniline. PANI can be easily detected by differential pulse voltammetry at a working voltage as low as 40 mV (vs. Ag/AgCl) which makes the assay fairly selective. This immunoassay for Cry1Ab has an analytical range from 0.1 to 1000 ng∙mL^-1 and a 0.07 ng∙mL^-1 lower limit of detection. The average recoveries of the toxin from spiked samples are in the range from 102 to 114 %, with a relative standard deviation of <7.5 %. The results demonstrated that the assay represented an attractive alternative to existing immunoassays in enabling affordable, sensitive, robust and specific determination of this toxin.

Nanobodies specific to Cry1Ab toxin were isolated from an immunized camel. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay with horseradish peroxidase being used to produce polyaniline, which can be easily detected by differential pulse voltammetry.

     

Parallel solution-phase synthesis of a 2,6,8,9-tetrasubstituted purine library via a sulfur intermediate

Purine analogues exhibiting a wide range of pharmacological activities have been considered a privileged structure in medicinal chemistry. In addition, the purine core consisting of four points of structural diversity is a well-sought scaffold in combinatorial chemistry. Although most of the efforts have been focused on 2,6,9-, 6,8,9-, or 2,8,9-trisubstituted purines, syntheses of 2,6,8,9-tetrasubstituted purines are rare. This paper presents a parallel solution phase approach for the synthesis of fully substituted purines via a 6-sulfur-substituted pyrimidine as the key intermediate. This strategy combining construction and modification of the purine ring thus increases the structural diversity of the final products. Sequential substitution of chlorines in 4,6-dichloro-2-methyl-5-nitropyrimidine with primary amine and benzylmercaptan afforded the 4-(substituted)amino-6-benzylthio-5-nitropyrimidine, which was readily converted to its diaminopyrimidine analogue by reduction of the nitro group. The diaminopyrimidine intermediate was cyclized to construct the purine ring with a C-8 substituent. Eventual oxidation of sulfur to sulfone and subsequent displacement by a primary or secondary amine provided the desired 2,6,8,9-tetrasubstituted purine analogues. This synthetic methodology was validated with the synthesis of a 216-member purine library.