猪β2m二级结构预测及同源模建

目的:研究猪β2m的结构.方法:应用EXPASY服务器(http://www.expasy.ch/tools)上的SOPMA法对β2m进行二级结构的预测,并与人的β2m的氨基酸和二级结构成分比较,在此基础上同源模建β2m的三级结构.结果:β2m二级结构成分α螺旋、β折叠、转角和无规则卷曲的数目分别为5、36、5和52,与人β2m相应二级结构的符合率分别达到100%(α螺旋)、92.3%(B折叠)、71.7%(转角)和92.3%(无规则卷曲),且二级结构的同源率要大于氨基酸的同源率.同源模建分析表明猪和人的3D结构也非常相似,只是存在个别氨基酸的差异.结论:猪和人β2m空间结构相似.     

聚α—氨基酸纤维的研究

<正> 在制造具有动物纤维相似性质的合成纤维时,应当引入蛋白质结构的慨念。丝是一种富含β-折叠结构的多肽,而羊毛则富有α-螺旋结构。当α-氨基酸聚合成聚α-氨基酸时,其一级结构与蛋白质一样为多肽,但其二级结构则可为α-螺旋、β-折叠或无规线圈,如果一种聚α-氨基酸纺成富含β-折叠     

α-天门冬氨酰二肽酶及其进化酶的FT-IR研究

用分子定向进化技术,在酶活力和热稳定性双重选择压力下,筛选到了一株Kcat/KM是天然酶47倍的进化酶.用FT-IR方法,测定了α-天门冬氨酰二肽酶及其进化酶的酰胺I带图谱,定量估算了天然酶和进化酶的各种二级结构含量.天然酶中,β折叠结构含量为28.5%,α螺旋结构含量为33%,这与园二色谱测量α螺旋结构为33%的结果有很好的一致,剩余的残基形成不同类型的转角和无规结构,其总含量为38.5%.在进化酶中,β折叠结构含量为26.8%,α螺旋结构含量为31%,其它结构为不同类型的转角和无规结构,含量为42.2%.     

川芎咖啡酸-3-O-甲基转移酶的同源建模与分子模拟对接

[目的]分析川芎咖啡酸-3-O甲基转移酶(Caffeic acid-3-Omethyltransferase,COMT)序列特征、蛋白结构和活性位点。[方法]以紫花苜蓿中COMT的晶体结构为模板,利用同源建模构建川芎(Ligusticum chuanxiong Hort)COMT的三维模型。[结果]经过动力学优化后,川芎COMT的三维模型与紫花苜蓿COMT的结构极为相似,主要由α-螺旋和β-折叠构成,其中α-螺旋占37.26%、β-折叠占10.41%、无规卷曲占52.33%。川芎COMT含有两个重要的结构域:咖啡酸结合域与S-腺苷甲硫氨酸结合域,主要通过氢键与范德华力结合,其中咖啡酸与川芎COMT分子中的Asp208和Gly210形成氢键,S-腺苷甲硫氨酸与川芎COMT分子中的Ser186、Thr213、Ala215、Thr216、Trp268及Asp272等残基形成氢键。[结论]该文得到川芎COMT的序列特征、蛋白结构和活性位点,为该类COMT的催化机理研究和分子工程改造奠定基础。     

用激光拉曼探讨不同相对湿度下聚赖氨酸分子的二级结构

本文报导了聚赖氨酸分子的二级结构因其含水量变化而变化.用T.L Lippert等人提出的方法对不同含水量下的聚赖氨酸分子的激光拉曼光谱进行了定量计算.结果表明分子二级结构中的x螺旋、β折叠及无规卷曲成份有明显差异.当分子中相对湿度在0～33%时它的二级结构主要为无规卷曲成份;当湿度在90%-75%时分子的主键盘方式以α螺旋为主;而当湿度小于75%大于40%范围时分子的二级结构改变以β折叠和无规卷曲的肽链构象.     

酸性和碱性酶稳定性机制及其识别

了解酸性和碱性酶稳定性机制并对其进行识别具有重要理论和实践意义。通过分析105条酸性酶和111条碱性酶序列的氨基酸组成, 结果表明: 酸性酶中Trp、Tyr、Thr和Ser的含量明显高于平均值, 而Glu、Lys、Met和Arg的含量则明显低于平均值; 碱性酶中Trp、Ala和Cys的含量略高于平均值, 而Lys、Arg和Glu的含量则略低于平均值; 酸性和碱性酶中Ala、Glu、Leu、Asn、Arg、Ser和Thr的含量存在较大差异。在此基础上, 发展了一种加权氨基酸组成的方法对两种酶进行识别, 其自一致性检验的识别精度可达86.1%, 5倍交叉验证的精度为83.3%。建立了一种基于序列识别酸性和碱性酶的新方法。     

东亚马氏钳蝎(Buthus martensi Karsch)神经毒素组分BmK Ⅲ的激光拉曼光谱研究

毒素BmKⅢ是从马氏钳蝎毒液中提取的一个毒性最强的α类神经毒素.从该组分的水溶液和重水溶液拉曼光谱(450—1750cm~(-1))的分析,可以确定其分子的二级结构中以β折迭为主,有较大量的β回折结构和一定数量的无规卷曲,α螺旋不多.采用Lippert提出的线性方程组法计算得到分子中含β折迭41.7%,α螺旋17.7%,无规卷曲40.6%.分子中的四对二硫键均为gauche—gauche—gauche构型.侧链芳香性氨基酸残基大多数暴露在分子表面.文中对有有关毒素构象方面的问题进行了讨论.     

东亚马氏钳蝎(Buthus martensi Karsch)神经毒素组分BmK Ⅲ的激光拉曼光谱研究

毒素BmKⅢ是从马氏钳蝎毒液中提取的一个毒性最强的α类神经毒素.从该组分的水溶液和重水溶液拉曼光谱(450—1750cm~(-1))的分析,可以确定其分子的二级结构中以β折迭为主,有较大量的β回折结构和一定数量的无规卷曲,α螺旋不多.采用Lippert提出的线性方程组法计算得到分子中含β折迭41.7%,α螺旋17.7%,无规卷曲40.6%.分子中的四对二硫键均为gauche—gauche—gauche构型.侧链芳香性氨基酸残基大多数暴露在分子表面.文中对有有关毒素构象方面的问题进行了讨论.     

耐热β-糖苷酶的基因克隆、表达和耐热性研究

从非解朊栖热菌HG10 2 (Thermusnonproteolyticus)染色体文库中 ,筛选得到耐热 β 糖苷酶 (Tn-gly)基因。该基因位于重组质粒 2.6kbHindⅢ插入片段上 ;并在E .coli中表达 ,酶比活力提高 17倍。经序列测定 ,该基因1311bp ,编码 436个氨基酸 ,前端与部分糖透性酶基因紧密相连 ,G+G含量为 71% ,有明显的RBS序列 ,启动子序列不明显。经序列同源性比较 ,其氨基酸序列属糖苷水解酶家族 1,有-N-E-P-和-T-E-N-保守序列 ,Glu16 4和Glu338推测是催化活性中心。其氨基酸组成中 ,疏水氨基酸含量较高 (Ala 12.8%、Leu 10.9% ) ,Arg(9.6% )、Glu(9.44 % )和Pro(8.0 % )含量显著较高。理论预测二级结构中 ,α 螺旋占 41.4% ,β-折叠占 16.2% ,β-转角占 14.4% ,并有大量的Pro位于β-转角的第二位。疏水作用、盐键、α-螺旋和Pro对Tn-gly的突出热稳定性有重要贡献。经系统进化树分析发现 ,β-糖苷酶在物种进化中比较保守。     

Ydj1p二聚体中β14~β15与domain-III分离的拉伸分子动力学模拟研究

分子伴侣Hsp40是一种以二聚体的形式调控非天然多肽折叠的热激蛋白。本文通过拉伸分子动力学研究了酵母Hsp40家族成员Ydj1p二聚体中β14-β15与domain-Ⅲ的分离过程,深入探讨了影响Ydj1p二聚体稳定性的重要残基和相互作用力。研究表明,残基Thr366、Asp368、Cys370、Leu372和Phe375在Ydj1P二聚体的形成过程中发挥着重要的作用。其中,β14-β15中的残基Thr366和Asp368分别通过与domain-Ⅲ内的残基Asp291、Trp292和Trp292、Lys294之间形成的氢键,Asp368通过与domain-Ⅲ内的残基Lys314形成盐桥,Cys370、Leu372和Phe375则是通过与domain-Ⅲ形成疏水作用力来稳定Ydj1p二聚体结构。     

Secondary structural changes in the intact and the disulfide Bridges cleaved β-lactoglobulin A and B in solutions of urea,guanidine hydrochloride,and sodium dodecyl sulfate

The relative proportions of α-helix, β-sheet, and unordered form in β-lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of β-lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% α-helix and 41% β-sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased β-sheet up to 48% but did not affect the α-helical proportion. The α-helical proportions of nonreduced β-lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the α-helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The β-sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Secondary structure perturbations in salt-induced protein precipitates

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na₂SO₄, were studied for twelve different proteins. α-helix and β-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of β-sheet structures with a concomitant loss of α-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of α-helix and β-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting β-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in α-helix content were influenced by the initial helical content and the dipole moment. The enhanced β-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.     

湖南尖吻蝮蛇毒出血毒素的圆二色性和化学修饰

用远紫外ＣＤ谱研究了湖南产尖吻蝮蛇毒的两个出血毒素（ＤａＨＴ－１、ＤａＨＴ－２）的溶液构象,计算得ＤａＨＴ－１的α螺旋、β折叠、无规卷曲的含量分别为３６．９％、３５．５％、２７．６％;ＤａＨＴ－２的α螺旋、β折叠、无规卷曲分别为２３．４％、３１．３％、４５．３％。随ｐＨ的增大或减小,峰位蓝移,酸性条件下的变化比碱性条件下的变化大。构象单元含量计算表明：α螺旋减少,无规卷曲增多,β折叠基本未变。温度和ｐＨ对ＣＤ谱的影响相似,５０℃时峰位蓝移,α螺旋减少,无规卷曲增多．ＥＤＴＡ对ＣＤ谱影响显著,０．０２ｍｏｌ／ＬＥＤＴＡ便导致两个出血毒素呈极度的无序状态。ＥＤＴＡ完全抑制,半胱氨酸部分抑制,胰蛋白酶不影响它们的出血活性。     

Flow-induced conformational changes and phase behavior of aqueous poly-L-lysine solutions

Poly-L -lysine exists as an α-helix at high pH and a random coil at neutral pH. When the α-helix is heated above 27°C, the macromolecule undergoes a conformational transition to a β-sheet. In this study, the stability of the secondary structure of poly-L -lysine in solutions subjected to shear flow, at temperatures below the α-helix to β-sheet transition temperature, were examined using Raman spectroscopy and CD. Solutions initially in the α-helical state showed time-dependent increases in viscosity with shearing, rising as much as an order of magnitude. Visual observation and turbidity measurements showed the formation of a gel-like phase under flow. Laser Raman measurements demonstrated the presence of small amounts of β-sheet structure evidenced by the amide I band at 1666 cm⁻¹. CD measurements indicated that solutions of predominantly α-helical conformation at 20°C transformed into 85% α-helix and 15% β-sheet after being sheared for 20 min. However, on continued shearing the content of β-sheet conformation decreased. The observed phenomena were explained in terms of a “zipping-up” molecular model based on flow enhanced hydrophobic interactions similar to that observed in gel-forming flexible polymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 239–246, 1998     

Circular dichroism and laser Raman spectroscopic analysis of the secondary structure ofCerebratulus lacteus toxin B-IV

The secondary structure ofCerebratulus lacteus toxin B-IV, a neurotoxic polypeptide containing 55 amino acid residues and four disulfide bonds, was experimentally estimated by computer analyses of toxin circular dichroism (CD) and laser Raman spectra. The CD spectrum of the toxin displayed typical α-helical peaks at 191, 208, and 222 nm. At neutralpH, the α-helix estimates from CD varied between 49 and 55%, when nonrepresentative spectrum analytical methods were used. Analysis of the laser Raman spectrum obtained at a much higher toxin concentration yielded a 78% α-helix estimate. Both CD and Raman spectroscopic methods failed to detect any β-sheet structure. The spectroscopic analyses revealed significantly more α-helix and less β-sheet for toxin B-IV than was predicted from its sequence. To account for the difference between the 49–55% helix estimate from CD spectra and the 78% helix estimate from the Raman spectrum, we postulate that some terminal residues are unfolded at the low toxin concentrations used for CD measurements but form helix at the high toxin concentration used for Raman measurements. Our CD observations showing thatCerebatulus toxin B-IV helix content increases about 15% in trifluoroethanol or at highpH are consistent with this interpretation.     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.     

The crystal structure of the dimeric colicin M immunity protein displays a 3D domain swap

Bacteriocins are proteins secreted by many bacterial cells to kill related bacteria of the same niche. To avoid their own suicide through reuptake of secreted bacteriocins, these bacteria protect themselves by co-expression of immunity proteins in the compartment of colicin destination. In Escherichia coli the colicin M (Cma) is inactivated by the interaction with the Cma immunity protein (Cmi). We have crystallized and solved the structure of Cmi at a resolution of 1.95? by the recently developed ab initio phasing program ARCIMBOLDO. The monomeric structure of the mature 10kDa protein comprises a long N-terminal α-helix and a four-stranded C-terminal β-sheet. Dimerization of this fold is mediated by an extended interface of hydrogen bond interactions between the α-helix and the four-stranded β-sheet of the symmetry related molecule. Two intermolecular disulfide bridges covalently connect this dimer to further lock this complex. The Cmi protein resembles an example of a 3D domain swapping being stalled through physical linkage. The dimer is a highly charged complex with a significant surplus of negative charges presumably responsible for interactions with Cma. Dimerization of Cmi was also demonstrated to occur in vivo. Although the Cmi-Cma complex is unique among bacteria, the general fold of Cmi is representative for a class of YebF-like proteins which are known to be secreted into the external medium by some Gram-negative bacteria.     

Design,synthesis, and conformation of a model peptide of endothelin with cystine-stabilized α-helix motif

A model 16-peptide of endothelin-1 (MET-1), which has the minimized sequence homology to the corresponding pan of endothelin-1 (ET-1), was designed to confirm the cystine-stabilized α-helix motif. The model structure consists of an extended structure, a β-turn part, and an α-helix structure that is stabilized by two disulfide bonds. The α-helix segment was designed to emphasize the amphiphilic nature. In order to combine the extended structure and the α-helix segment, a D -Ala-Pro sequence was selected to fix the β-turn. The model endothelin 16-peptide amide was synthesized by solid-phase synthesis on a 4-methylbenzhydrylamine resin. Its conformation was examined by CD and two-dimensional (2D) ¹H-nmr measurements. MET-1 showed similar CD patterns to ET-1 in both buffer and 50% aqueous trifluoroethanol solution. The 2D nmr experiments in 50% aqueous ethylene glycol revealed that MET-1 closely resembles the conformation of ET-1 with an extended structure, an α-helix, and a β-turn unit in the same position of the sequence. Furthermore, model peptides without disulfide bond(s) could not assume a stable structure in aqueous solution, while they did have similar α-helical content in 50% trifluoroethanol with MET-1. When the two disulfide bridges were simultaneously formed, the peptide with the correct disulfide bonds (MET-1) was obtained in threefold excess to the isomer (apamin type. MET-2). These findings obtained by the modeling of ET-1 showed an important role for the stabilization of peptide conformation with disulfide bonds. © 1994 John Wiley & Sons, Inc.     

Hydrogen-bond cooperativity in protein secondary structure

Hydrogen bonding in the α-helix and β-sheet has been studied by ab initio molecular orbital calculations carried out on complexes of formamide. Hydrogen-bond geometries were taken from x-ray crystallography of polypeptides. Positive cooperativity is found in all cases. The limiting value for infinite chains is obtained by use of a double-reciprocal plot and indicates an increase in the effective bond strength of 25% over that of a single isolated bond. Parallel calculations based on a classical electrostatic model yield qualitatively similar trends but underestimate the cooperativity by half. Charge redistribution accompanying cooperativity is characterized by a new type of charge-density difference plot, the cooperativity map. The magnitude and distance over which cooperativity acts suggest several significant biological consequences. Thus the average of α-helices and the number of β-sheet strands found in protein may be influenced by cooperativity. Cooperativity in the interpeptide hydrogen bond may also be partly responsible for the rapid formation of secondary structure in renaturing proteins and help stabilize secondary structure relative to the random-coil conformation.     

Conformational transitions of calmodulin as studied by vacuum-uv CD

CD measurements were made for calmodulin and its calcium (Ca²⁺) complexes at different ionic strengths and Ca²⁺ concentrations. Calmodulin at an ionic strength of 0.00M and in the absence of Ca²⁺ exists as an α-helical protein with a negligible amount of β-sheet. An increase in ionic strength, whether or not Ca²⁺ is present, increases α-helix at the expense of “other” (coil) structure. The changes in β-sheet and β-turns are insignificant. Binding of Ca²⁺ at low ionic strength occurs in stages with at least one folding intermediate before attaining the final stable state. Binding of Ca²⁺ at an ionic strength of 0.165M causes only a slight increase in α-helix, so that the secondary structure of the protein depends on ionic strength and is insensitive to the nature of the cation (i.e., Ca²⁺). Thus, the activation of calmodulin by Ca²⁺ must be due to a structural reorientation rather than to a major secondary structural alteration. The CD estimation of secondary structure with 4 mol Ca²⁺/calmodulin (61% α-helix, 2% antiparallel β-sheet, 2% parallel β-sheet, 21% β-turns, and 14% other) is in excellent agreement with the x-ray results.