链霉菌降解角蛋白的生化机制研究*

对弗氏链霉菌S-221变种降解角蛋白的生化机制进行了初步研究。该菌在角蛋白底物作用下诱导产生角蛋白酶。它是一种复合蛋白酶,含有二硫键还原酶和多肽水解酶等多种酶活性组分。硫酸钠、亚硫酸钠和巯基乙醇对角蛋白酶具有强烈的激活作用,其主要表现作用于角蛋白酶中的二硫键还原酶。亚硫酸钠在0·01mol/L浓度下不仅作用于二硫键还原酶,而且还作用于多肽水解酶。硫代硫酸钠对二硫键还原酶有强烈的抑制作用。角蛋白酶降解羽毛角蛋白首先是角蛋白酶中的二硫键还原酶使角蛋白中二硫键裂解产生变性角蛋白,然后变性角蛋白在多肽水解酶的共同     

链霉菌降解角蛋白的生化机制研究

对弗氏链霉菌S-221变种降解角蛋白的生化机制进行了初步研究。该菌在角蛋白底物作用下诱导产生角蛋白酶。它是一种复合蛋白酶,含有二硫键还原酶和多肽水解酶等多种酶活性组分。硫酸钠、亚硫酸钠和巯基乙醇对角蛋白酶具有强烈的激活作用,其主要表现作用于角蛋白酶中的二硫键还原酶。亚硫酸钠在0．01mol／L浓度下不仅作用于二硫键还原酶,而且还作用于多肽水解酶。硫代硫酸钠对二硫键还原酶有强烈的抑制作用。角蛋白酶降解羽毛角蛋白首先是角蛋白酶中的二硫键还原酶使角蛋白中二硫键裂解产生变性角蛋白,然后变性角蛋白在多肽水解酶的共同作用下逐步水解成多肽、寡肽和游离氨基酸,使角蛋白彻底降解。在角蛋白降解过程中,角蛋白中的硫也随之转化成巯基化合物,H2S和硫酸盐3种含硫化合物存在于降解产物中。     

崔大敷研究员关于催产素工作的几点体会

<正>催产素是一种由9个氨基酸残基组成的多肽类激素,分子内形成1对二硫键,适用于产妇子宫收缩,产后止血。催产素原来的生产工艺是从猪脑垂体后叶中提取。1953年,美国化学家维格奥纳德(Vincent du Vigneand)等人合成了第一个天然多肽激素——含有二硫键的催产素,找到了将二硫键引入多肽分子的一条途径,并因此获得了1955年的诺贝尔化学奖。1958年7月,当人工合成结晶牛     

重组单链胰岛素在含有巯基试剂的变性剂中的解折叠

重组单链胰岛素(PIP)含有3对二硫键。在含有巯基试剂的变性剂中,PIP产生二硫键交换从而形成一系列具有不同解折叠程度的二硫键异构体混合物。分别用高压液相色谱（HPLC）和圆二色性（CD）光谱分析了PIP在含有0．2mmol/L2－巯基乙醇的尿素和盐酸胍中的解中的解折叠程度。PIP二硫键异构体混合物通过胰蛋白酶酶解并用质谱测定酶解片段的分子量,证明PIP确实产生了二硫键交换。同时还分离纯化了PIP的一种主要非天然二硫键异构体并研究了它重新折叠成天然构象的情况。观察到PIP只有一种热力学稳定的二硫键配对方式,PIP的非天然二硫键异构体在巯基试剂存在的条件下可以高效转化为天然二硫键配对。还将PIP解折叠和再折叠的情况与胰岛素样生长因子－I（IGF－I）及胰岛素做了比较：胰岛素和PIP只折叠成一种热力学稳定的三级结构,IGF－I却折叠成两种热力学稳定的二硫键异构体;胰岛素的双链重组需缓慢进行,而PIP却可以快速折叠。     

从噬菌体多肽文库中筛选α—葡萄糖苷酶的抑制剂

以黑曲霉葡糖淀粉酶为靶分子,筛选噬菌体展示的多肽文库,经过3轮生物淘选法筛选,得到3个特异性结合的噬菌体克隆,分别命名为GB-1、GB2和GB-3。其插入多肽中都含有一对二硫键,破坏二硫键将大大降低噬菌体与葡糖淀粉酶的结合。化学合成的环状多肽KCHFEECLAY,其序列对应于结合力最强的一个克隆GB-1的N端的10个氨基酸残基,该多肽可以竞争性地抑制黑曲霉葡糖淀粉酶（Ki=0.2mmol/L)以及大鼠小肠的α-葡萄糖苷酶（Ki=1.4mmol/L)。     

新型α-芋螺毒素Di1.1的克隆、合成及功能研究

目的：从中国南海长距芋螺中克隆出新的芋螺毒素序列并用固相方法合成该毒素,测定其折叠后的二硫键配对方式并初步研究其药理学特性。方法：根据芋螺毒素A超家族保守的信号肽序列设计引物,通过3＇-RACE扩增,从芋螺毒腺管中克隆出新的毒素基因;采用Fmoc-固相法合成线性多肽,通过空气氧化折叠获得含二硫键的折叠产物,用两步氧化折叠法测定多肽的二硫键连接方式;用双电极电压钳技术初步研究其药理学特性。结果：发现-种新的α-芋螺毒素Dil．1的cDNA序列,其成熟肽序列为CcVIESCHSNHIDECES;该肽二硫键连接方式以C1-C4、C2-C3为主,以C1-C3、C2-C4连接为辅,对烟碱型乙酰胆碱受体各亚型活性较弱。结论：Dil．1是-种新的α4／7型芋螺毒素,其折叠方式以C1-C4、C2-C3连接为主。     

毛细管电泳测定牛红细胞超氧化物歧化酶的巯基位点

本文介绍了毛细管电泳分析蛋白质酶解产物中含巯基多肽的方法。还原的及天然的牛红细胞超氧化物歧化酶(SOD)经4-乙烯吡啶修饰后,由TPCK-胰蛋白酶水解,在254nm检测到还原的SOD水解物中含3个巯基多肽,天然的SOD为1个疏基多肽且其毛细管电泳行为与上述3个多肽之一相一致。分析它们的氨基酸顺序,证实Cys-6为游离的巯基,Cys-55和Cys~(-144)形成二硫键。     

虎纹捕鸟蛛毒素V的二硫键定位分析

虎纹捕鸟蛛毒素V是从虎纹捕鸟蛛毒液中分离得到的一种昆虫毒素．它含有35个氨基酸残基,其中6个半胱氨酸形成三对二硫键．首先采用多酶将天然的肽链裂解后,通过MALDI-TOF质谱分析酶解肽段,推断出1对二硫键位于Cys9-Cys21,然后利用改进的部分还原分步测序法,确定虎纹捕鸟蛛毒素V的另外2对二硫键的配对方式为Cys2-Cysl6和Cys15-Cys28．因此,虎纹捕鸟蛛毒素V的3对二硫键分别以Cys2-Cys16,Cys9一Cys21,Cys15一Cys28(即1-4、2-5和3-6)的方式配对．     

虎纹捕鸟蛛毒素Ⅴ的二硫键定位分析

虎纹捕鸟蛛毒素Ⅴ是从虎纹捕鸟蛛毒液中分离得到的一种昆虫毒素.它含有35个氨基酸残基,其中6个半胱氨酸形成三对二硫键.首先采用多酶将天然的肽链裂解后,通过MALDI-TOF质谱分析酶解肽段,推断出1对二硫键位于Cys9-Cys21,然后利用改进的部分还原分步测序法,确定虎纹捕鸟蛛毒素Ⅴ的另外2对二硫键的配对方式为Cys2-Cys16和Cys15-Cys28.因此,虎纹捕鸟蛛毒素Ⅴ的3对二硫键分别以Cys2-Cys16,Cys9-Cys21,Cys15-Cys28（即1-4、2-5和3-6）的方式配对.     

抑制剂胱氨酸结多肽的抗菌和杀虫活性及进化

抑制剂胱氨酸结(inhibitor cystine knot, ICK)多肽是胱氨酸结(cystine knot, CK)多肽的三大家族成员之一。这种类型的多肽含有反向平行的3个β折叠,由3对二硫键形成稳定的拓扑学打结。它们广泛地分布在动物、植物、真菌甚至原核生物中,具有蛋白酶抑制剂,离子通道毒素以及抗微生物、抗疟疾及抗病毒活性等多种生物学功能。本文首先总结了昆虫中已发现的抗微生物活性和离子通道毒素活性的ICK肽;然后介绍了有毒动物尤其是蜘蛛、蝎子,以及植物中一些神经毒素活性的ICK肽,它们通常靶向昆虫体内的各种离子通道,从而发挥杀虫效果;最后结合ICK肽的基因序列特征,结构域和二硫键数目以及物种分布,对其进化多样性进行了探讨。     

The contribution of cross-links to protein stability: A normal mode analysis of the configurational entropy of the native state

The vibrational entropy of native BPTI, with three disulfide bonds, was determined by use of normal mode calculations and compared with that of folded variants having either one less disulfide bond or lacking a peptide bond at the trypsin-reactive site. Favorable contributions to the free energy of 2.5–5.1 kcal/mol at 300 K were calculated for the reduction of disulfide bonds in the folded state, whereas no favorable contribution was found for the hydrolysis of the peptide bond cleaved by trypsin. This is on the order of the effect of disulfides in the unfolded state. The implications of these results for the stabilization of a folded protein by the introduction of crosslinks are discussed. © 1993 Wiley-Liss, Inc.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

红细胞生长刺激蛋白二硫键定位连接方式的测定

目的:建立红细胞生长刺激蛋白(ESP)的二硫键连接方式的测定方法。方法:先将红细胞生长刺激蛋白的糖链用糖苷酶切除,再分别对ESP和还原烷基化后ESP用胰蛋白酶进行酶切,然后用MALDI-TOF测得该蛋白质的肽质量指纹图谱,通过比较还原烷基化前后各肽质量指纹图谱,找到差异肽段的分子离子峰[M+H]+,通过比对该蛋白理论酶切肽的[M+H]+,确定二硫键的连接方式。结果:ESP有4个半胱氨酸,通过比较还原烷基化前后的肽质量指纹图谱定位二硫键的位置为Cys7-Cys161和Cys29-Cys33,与理论上的二硫键连接相符。结论:建立了酶切结合质谱法测定蛋白质二硫键定位的方法,为今后生物技术产品的二硫键连接方式的质量控制提供了有效的方法。     

Purification of correctly oxidized MHC class I heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding

The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC-I) heavy-chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy-chain molecules in bacteria results in the formation of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and refolding is complicated by the fact that (1). correct folding can only take place in combined presence of beta(2)-microglobulin and a binding peptide; and (2). optimal in vitro conditions for disulfide bond formation ( approximately pH 8) and peptide binding ( approximately pH 6.6) are far from complementary. Here we present a two-step strategy, which relies on uncoupling the events of disulfide bond formation and peptide binding. In the first phase, heavy-chain molecules with correct disulfide bonding are formed under non-reducing denaturing conditions and separated from scrambled disulfide bond forms by hydrophobic interaction chromatography. In the second step, rapid refolding of the oxidized heavy chains is afforded by disulfide bond-assisted folding in the presence of beta(2)-microglobulin and a specific peptide. Under conditions optimized for peptide binding, refolding and simultaneous peptide binding of the correctly oxidized heavy chain was much more efficient than that of the fully reduced molecule.     

Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern

La1 is a 73‐residue cysteine‐rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N‐acylurea approach with Fmoc‐SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of peptide substrates for mammalian thioredoxin reductase.

Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form eight-membered ring structures and are difficult to synthesize owing to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers (Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174-177). First, the addition of an amine base to the deprotection solution allows the complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide-linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring.     

杂环肽FIK的Fmoc固相合成法研究

为研究二硫键成环的杂环肽FIK的合成工艺, 以Fmoc氨基酸为原料, 采用固相合成法, 经TBTU/HOBT/DIEA复合缩合剂催化合成直链肽, 再经I2氧化肽链上两个半胱氨酸的巯基生成分子内二硫键而得到目标环肽, 将其用切割试剂切割脱离树脂得到粗产品, MALDI-MS和RP-HPLC进行鉴定, 分析和纯化。产率可以达到18%, 纯化后纯度达97%以上, 经MALDI-MS和Ellman试剂检测确定为目标肽。该合成法高效, 简便, 快速, 目标肽收到较理想产率, 适合大批量生产。     

A novel methodology for assignment of disulfide bond pairings in proteins.

A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.     

Chemical synthesis of N‐glycosylated insulin‐like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii

Crustacean insulin‐like androgenic gland factor (IAG) of Macrobrachium rosenbergii, a heterodimeric peptide having both four disulfide bonds and an N‐linked glycan, was synthesized by the combination of solid‐phase peptide synthesis and the regioselective disulfide formation reactions. The disulfide isomer of IAG could also be synthesized by the same manner. The conformational analysis of these peptides by circular dichroism (CD) spectral measurement indicated that the disulfide bond arrangement affected the peptide conformation in IAG. On the other hand, the N‐linked glycan attached at A chain showed no effect on CD spectra of IAG. This is the first report for the chemical synthesis of insulin‐like heterodimeric glycopeptide having three interchain disulfides, and the synthetic strategy shown here might be useful for the synthesis of other glycosylated four‐disulfide insulin‐like peptides.     

Structure-activity relationships of the intramolecular disulfide bonds in coprisin,a defensin from the dung beetle

Defensins, which are small cationic molecules produced by organisms as part of their innate immune response, share a common structural scaffold that is stabilized by three disulfide bridges. Coprisin is a 43-amino acid defensin-like peptide from Copris tripartitus. Here, we report the intramolecular disulfide connectivity of cysteine-rich coprisin, and show that it is the same as in other insect defensins. The disulfide bond pairings of coprisin were determined by combining the enzymatic cleavage and mass analysis. We found that the loss of any single disulfide bond in coprisin eliminated all antibacterial, but not antifungal, activity. Circular dichroism (CD) analysis showed that two disulfide bonds, Cys20-Cys39 and Cys24-Cys41, stabilize coprisin’s α-helical region. Moreover, a BLAST search against UniProtKB database revealed that coprisin’s α-helical region is highly homologous to those of other insect defensins. [BMB Reports 2014; 47(11): 625-630]