肝素诱导人细胞型朊蛋白构象变化的光谱表征

粘多糖在朊病毒病中所发挥的作用目前仍存在争议.以肝素钠作为粘多糖的代表,通过共振光散射光谱、荧光光谱和圆二色光谱的变化研究了肝素钠与人重组细胞型朊蛋白(rhPrPC23-231)的相互作用.结果表明,肝素钠与朊蛋白相互作用后光散射和荧光信号均得到增强,并且使朊蛋白的荧光寿命有一定程度的延长.圆二色光谱表明肝素钠能诱导朊蛋白从富含α-螺旋的构象向富含β-折叠的构象转变.     

功能肽段介导的铜离子荧光传感器的构建及应用

铜离子是导致多种疾病(如阿尔茨海默病、朊病毒病和肌萎缩性侧索硬化症等)的重要因素.当前已有针对铜离子检测的相关报道,但基于多肽与铜离子相互作用的荧光传感器鲜有关注.该文依据淀粉样蛋白与铜离子特异性结合的特点,利用已知的淀粉样蛋白组成单元,设计出能与铜离子有序配位的多肽分子(DDAEGHARHCR).同时,借助于"多肽-...     

人类朊蛋白的动力学稳定性研究（英文）

朊蛋白病是一种能在人类或者动物之间传播的致命的神经退行性疾病.尤其是人类朊蛋白疾病在近几年蔓延迅速,已经威胁到人类的健康.在本文中,我们使用分子动力学(MD)和流体分子动力学(FMD)模拟相结合的方法研究了人类朊蛋白(hPrPc)的动力学稳定性.我们通过FMD模拟产生了两个典型的hPrPc的变性结构,并进一步研究了在自然状态下这两个变性结构重折叠的过程,从关键残基、二级结构、残基-残基相互作用图等方面详细讨论了hPrPc的解折叠和重折叠路径.研究发现hPrPc的三个α-螺旋结构组成了一个疏水核心,在蛋白质的解折叠和重折叠过程中发挥了重要的作用.刚性的疏水核心就像是脚手架一般为hPrPc的重折叠提供便利.在重折叠过程中,π-螺旋和310螺旋出现几率较高,并且β-折叠的延长也更多地出现在完全解折叠的hPrPc体系中.     

兔子朊蛋白结构稳定性的分子动力学研究(英文)

朊蛋白病是一种能够对人类和动物带来致命影响,并具有高度传染性的神经退行性疾病.兔子是目前已经报道的哺乳类动物中对朊蛋白病免疫的少数几个物种之一.我们将分子动力学和操控式分子动力学模拟相结合,研究了兔子正常朊蛋白的结构稳定性;同时讨论了蛋白结构的收敛性及刚性分布,并揭示了兔子朊蛋白中关键二级结构的动力学以及受力各向异性特征,证实了兔子朊蛋白结构的稳定性特征.     

脱氢氨基酸的合成及其在药物研发中的应用

传统多肽所具有的容易被酶解、细胞膜通透性差以及构象容易发生变化等缺点,限制了它作为药物在疾病治疗领域的应用。将脱氢氨基酸引入多肽,对其进行构象限制,能够有效改善它的代谢稳定性和生物利用度。本文主要综述了α,β-脱氢-α-氨基酸、β,γ-脱氢-α-氨基酸、α-脱氢-β-氨基酸、α,β-脱氢-β-氨基酸四种脱氢氨基酸的合成方法以及近几年来在药物设计中的应用,希望为相关的研究提供参考。     

谷朊蛋白的改性及其作为纸张增强剂的性能

采用廉价可生物降解的小麦谷朊蛋白为原料,经过羟甲基化和阳离子化改性合成类似聚酰胺聚胺环氧氯丙烷(PPE)的纸张增强剂. 经改性后,谷朊蛋白带有氮杂环丁烷结构、表氯醇和环氧基团3种功能基团,可与纤维形成共价键,且能发生自身交联,在纤维周围形成三维交联网络结构,提高纸张干、湿强度. 通过单因素试验,研究了甲醛、甲酸、温度、反应时间和环氧氯丙烷5种反应因素对纸张强度的影响. 优化合成条件下制备改性谷朊蛋白可使纸张干抗张强度提高35%,湿强保留率达20%. 改性后谷朊蛋白显阳离子性,加入纸浆中,可使得浆料体系Zeta电位升高,改善浆料的留着率,明显提高纸张强度. 结果表明,经羟甲基化和环氧氯丙烷加成改性的谷朊蛋白可以作为纸张的干强剂和湿强剂.     

麋鹿朊蛋白结构稳定性的分子动力学研究

朊蛋白病是一种致命且具有高度传染性的神经退行性疾病.糜鹿是目前已经报道的哺乳类动物中较易发生朊蛋白病的物种之一.作者使用分子动力学和操控式分子动力学模拟相结合的方法对糜鹿正常朊蛋白的结构稳定性进行了研究.发现了麋鹿朊蛋白结构中的不稳定结构域分布以及热动力学性质,揭示了糜鹿朊蛋白稳定性的分子结构基础以及力学特征.     

牡蛎源类蛋白反应修饰肽的分离纯化及肽锌螯合物的结构表征

以牡蛎为原料制备了类蛋白反应修饰肽,利用Sephadex G-15凝胶层析柱和反向高效液相色谱(RP-HPLC)等分离技术得到1条锌离子螯合活性为161 mg/g的多肽(M_w=835),多肽序列为EVPPEEH.以测得的肽序列为模板合成多肽,将纯肽与锌离子进行螯合反应制备肽锌螯合物.螯合物的红外光谱和圆二色光谱表征结果表明,锌离子主要与多肽链上的羰基氧发生相互作用.与多肽的空间结构相比,螯合物的无规则卷曲结构减少,β转角增加而β折叠减少.由肽锌螯合物的分子模拟和二级质谱结果可知,多肽与锌离子螯合后有2种空间构象:一种通过六配位的方式螯合1个锌离子,其中主要的螯合位点为多肽Val-2和Pro-3或者Glu-5和Glu-6之间的羰基氧;另一种是通过四配位的方式螯合1个锌离子,主要的螯合位点为多肽Glu-5和Glu-6之间的羰基氧.     

磺化酞菁与人血清白蛋白复合物的光动力活性研究

通过光谱法研究了三种磺化酞菁(α位四磺化酞菁、β位四磺化酞菁和α位单取代磺化酞菁)与人血清白蛋白(HSA)的相互作用.结果 表明,HSA对α位四磺化酞菁的存在状态(单体、聚集体)影响显著,而对β位四磺化酞菁和α位单取代磺化酞菁的存在状态没有明显影响.磺化酞菁与HSA均存在明显的相互作用,且.四磺化酞菁与HSA的结合作用...     

海藻糖抑制淀粉质多肽42构象转变的分子动力学模拟

应用分子动力学模拟方法研究了海藻糖抑制淀粉质多肽42(Aβ42)构象转变的分子机理.结果表明,海藻糖溶液浓度对Aβ42构象转变具有非常重要的影响.在水和低浓度海藻糖溶液(0.18mol·L-1)中,Aβ42可由初始的α-螺旋结构转变成β-折叠的二级结构;但海藻糖浓度为0.37mol·L-1时即可有效抑制Aβ42的构象转变.这是因为海藻糖利用其优先排阻作用使水分子在多肽周围0.2nm内富集,而其自身却在距离多肽0.4nm的位置附近团聚.另外,海藻糖还可通过降低多肽间的疏水相互作用,减少多肽分子内远距离的接触,有效抑制多肽的疏水塌缩和构象转变.上述分子模拟的结果对于进一步合理设计阿尔茨海默病的高效抑制剂具有非常重要的理论指导意义.     

Discriminant analysis of prion sequences for prediction of susceptibility

Prion diseases, including ovine scrapie, bovine spongiform encephalopathy (BSE), human kuru and Creutzfeldt–Jakob disease (CJD), originate from a conformational change of the normal cellular prion protein (PrP^C) into abnormal protease-resistant prion protein (PrP^Sc). There is concern regarding these prion diseases because of the possibility of their zoonotic infections across species. Mutations and polymorphisms of prion sequences may influence prion-disease susceptibility through the modified expression and conformation of proteins. Rapid determination of susceptibility based on prion-sequence polymorphism information without complex structural and molecular biological analyses may be possible. Information regarding the effects of mutations and polymorphisms on prion-disease susceptibility was collected based on previous studies to classify the susceptibilities of sequences, whereas the BLOSUM62 scoring matrix and the position-specific scoring matrix were utilised to determine the distance of target sequences. The k-nearest neighbour analysis was validated with cross-validation methods. The results indicated that the number of polymorphisms did not influence prion-disease susceptibility, and three and four k-objects showed the best accuracy in identifying the susceptible group. Although sequences with negative polymorphisms showed relatively high accuracy for determination, polymorphisms may still not be an appropriate factor for estimating variation in susceptibility. Discriminant analysis of prion sequences with scoring matrices was attempted as a possible means of determining susceptibility to prion diseases. Further research is required to improve the utility of this method.     

氯化钠对朊病毒结构稳定性影响的分子动力学研究

朊病毒疾病是由正常构象的PrPC转化为致病构象的PrPSc引起的一类可传染的蛋白质构象病.采用分子动力学模拟的方法研究了0～500mmol/L的NaCl溶液体系对人朊病毒构象影响并深入探讨了其分子机制.研究发现NaCl可以降低朊病毒的结构稳定性,并引起其α-螺旋含量的急剧降低.进一步的研究表明高浓度NaCl溶液体系能够显著破坏朊病毒螺旋1内部的重要盐桥Asp144-Arg148和Asp147-Arg151,同时明显降低其主要氢键Arg151 N:Asp147 O,Tyr150 N:Glu146 O,Tyr149 N:Tyr145 O和Arg148 N:Asp144 O的稳定性,并诱导朊病毒的疏水核心发生明显扩张,促使朊病毒整体稳定性的下降,这些可能是NaCl促进朊病毒构象转换的重要原因.     

Genetic algorithms as a tool for helix design – computational and experimental studies on prion protein helix 1

Summary Evolutionary computing is a general optimization mechanism successfully implemented for a variety of numeric problems in a variety of fields, including structural biology. We here present an evolutionary approach to optimize helix stability in peptides and proteins employing the AGADIR energy function for helix stability as scoring function. With the ability to apply masks determining positions, which are to remain constant or fixed to a certain class of amino acids, our algorithm is capable of developing stable helical scaffolds containing a wide variety of structural and functional amino acid patterns. The algorithm showed good convergence behaviour in all tested cases and can be parameterized in a wide variety of ways. We have applied our algorithm for the optimization of the stability of prion protein helix 1, a structural element of the prion protein which is thought to play a crucial role in the conformational transition from the cellular to the pathogenic form of the prion protein, and which therefore poses an interesting target for pharmacological as well as genetic engineering approaches to counter the as of yet uncurable prion diseases. NMR spectroscopic investigations of selected stabilizing and destabilizing mutations found by our algorithm could demonstrate its ability to create stabilized variants of secondary structure elements.     

朊病毒和疯牛病中蛋白自由基化学问题的探讨

２０年来,人们一直认为朊蛋白病变是导致疯牛病的原因,但对其致病机制却一直未得出公认的结论。其中的“蛋白错折叠”学说被多数人所接受,然而无法解释病中的多菌株现象,在以往的研究中,朊蛋白病变从来没有与任何化学问题有过联系。近年,我们对哺乳动物所具有的此类脑神经疾病进行分析,认为蛋白氧化损伤所形成的序列专一的长寿命朊蛋白自由基所催化下的蛋白氧化交联,可能是致病的根本原因。     

The yin and yang of amyloid: insights from α-synuclein and repeat domain of Pmel17

Amyloid has been traditionally viewed in the context of disease. However, the emerging concept of 'functional amyloid' has taken a new direction into how we view amyloid. Recent studies have identified amyloid fibrils ranging from bacteria to humans that have a beneficial role, instead of being associated with a misfolded state that has been implicated in diseases such as Alzheimer's, Parkinson's and prion diseases. Here, we review our work on two human amyloidogenic polypeptides, one associated with Parkinson's disease, α-synuclein (α-syn), and the other important for melanin synthesis, the repeat domain (RPT) from Pmel17. Particularly, we focused our attention on spectroscopic studies of protein conformation and dynamics and their impact on α-syn amyloid formation and for RPT, we discussed the strict pH dependence of amyloid formation and its role in melanin biosynthesis.     

Efficient Synthesis of Methanesulphonate-Derived Lipid Chains for Attachment of Proteins to Lipid Membranes

We have developed an easy and flexible synthetic methodology to obtain lipid chains containing methanothiosulfonate terminal groups with the aim to attach them to natural proteins as functional groups. There are many proteins found in nature that are modified by lipids, and this is a key part of their function. For example, the prion protein is attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor, and this protein is thought to be the causative agent in diseases such as bovine spongiform encephalopathy (BSE; “mad cow disease”) and the human equivalent Creutzfeldt–Jakob disease. However, production of large amounts of protein in bacteria results in proteins that lack these lipid modifications. The lipid chains containing methanothiosulfonate terminal groups that we have synthesized here can be attached to these proteins through the thiol contained in the side chain of the cysteine residue, which can be incorporated into the protein sequence at the desired position.     

Absolute Quantification of Prion Protein (90–231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

Substantial evidence indicates that the disease-associated conformer of the prion protein (PrP(TSE)) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrP(TSE) has the ability to convert the conformation of the normal prion protein (PrP(C)) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrP(TSE) at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrP(TSE), the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.     

High‐resolution differentiation of transmissible spongiform encephalopathy strains by quantitative N‐terminal amino acid profiling (N‐TAAP) of PK‐digested abnormal prion protein

New forms of transmissible spongiform encephalopathy (TSE) continue to be identified, and consequently sensitive differential diagnosis is increasingly important both for the management of disease in humans and livestock and in providing confidence in the safety of the food chain. TSE diseases are associated with accumulation of protease‐resistant prion protein (PrP^Sc) and detection of this marker protein is central to diagnosis. Proteolysis by proteinase K (PK) generates protease‐resistant products (PrP^res) with partially variable N‐termini. The conformation(s) of PrP^Sc and thus the points of PK cleavage are thought to be dependent on the strain of prion disease. Western blot (WB) analysis of PrP^res gives characteristic migration patterns that can be used to diagnose TSEs, but the relatively low resolution of this technique limits its ability to differentiate certain disease strains. Mass spectrometry (MS) has the capability to resolve these various PK cleavage sites to the level of individual amino acid residues. In the present study multiple selected reaction monitoring (mSRM) was used to detect and quantify PrP^res N‐terminal tryptic peptides by MS and thus to define the N‐terminal amino acid profiles (N‐TAAPs) of PrP^res characteristic for various TSEs in sheep. The fragmentation behaviour of the N‐terminal tryptic peptides was studied to allow selection of the transitions specific for each peptide. Different PrP^res preparation methods were evaluated and the most effective approach applied to differentiate the N‐TAAPs corresponding to various sheep TSE isolates. Marked differences were identified between the N‐TAAPs of bovine spongiform encephalopathy (BSE) and classical scrapie, and between classical scrapie and the experimental strains SSBP/1 and CH1641, thereby validating this approach as a means of TSE‐strain specific diagnosis. Copyright © 2008 John Wiley & Sons, Ltd.