蛋白质折叠类型的分类建模与识别

蛋白质的氨基酸序列如何决定空间结构是当今生命科学研究中的核心问题之一. 折叠类型反映了蛋白质核心结构的拓扑模式, 折叠识别是蛋白质序列-结构研究的重要内容. 我们以占Astral 1.65序列数据库中α, β和α/β三类蛋白质总量41.8%的36个无法独立建模的折叠类型为研究对象, 选取其中序列一致性小于25%的样本作为训练集, 以均方根偏差(RMSD)为指标分别进行系统聚类, 生成若干折叠子类, 并对各子类建立基于多结构比对算法(MUSTANG)结构比对的概形隐马尔科夫模型(profile-HMM). 将Astral 1.65中序列一致性小于95%的9505个样本作为检验集, 36个折叠类型的平均识别敏感性为90%, 特异性为99%, 马修斯相关系数(MCC)为0.95. 结果表明: 对于成员较多, 无法建立统一模型的折叠类型, 基于RMSD的系统分类建模均可实现较高准确率的识别, 为蛋白质折叠识别拓展了新的方法和思路, 为进一步研究奠定了基础.     

Recognition of Staphylococcus enterotoxin via molecularly imprinted beads

The synthesis of molecularly imprinted beads for the recognition of the protein Staphylococcus enterotoxin B (SEB) is described. Two kinds of organic silane (3-aminopropyltrimethoxysilane (APTMS) and octyltrimethoxysilane (OTMS)) were polymerized on the surface of polystyrene microspheres after the SEB template was covalently immobilized by forming imine bonds. The resulting imprinted beads were selective for SEB. The Langmuir adsorption models were applied to describe the equilibrium isotherms. The results showed that an equal class of adsorption was formed in the molecularly imprinted polymer (MIP) with the maximum adsorption capacity of 3.86 mg SEB/g imprinted beads. The MIP has much higher adsorption capacity for SEB than the nonimprinted polymer, and the MIP beads have a higher selectivity for the template molecule.     

Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners

Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell‘s hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell‘s environment and consequent protein expression profile.     

小波神经网络在紫外光谱识别中的应用研究

介绍用于信号识别的小波神经网络的结构和算法，并将其用于酪氨酸，二羟基苯丙氨酸和色氨酸的紫外光谱识别。在波小神经网络中，采用Ｍｏｒｌｅｔ母波波和一维搜索变步长共轭梯度优化方法。结果表明，小波神经网络对于光谱间的细微结构差别具有很好的识别能力。     

Combined Chemical Modification and Collision Induced Unfolding Using Native Ion Mobility-Mass Spectrometry Provides Insights into Protein Gas-Phase Structure

Native mass spectrometry is now an important tool in structural biology. Thus, the nature of higher protein structure in the vacuum of the mass spectrometer is an area of significant interest. One of the major goals in the study of gas-phase protein structure is to elucidate the stabilising role of interactions at the level of individual amino acid residues. A strategy combining protein chemical modification together with collision induced unfolding (CIU) was developed and employed to probe the structure of compact protein ions produced by native electrospray ionisation. Tractable chemical modification was used to alter the properties of amino acid residues, and ion mobility-mass spectrometry (IM-MS) utilised to monitor the extent of unfolding as a function of modification. From these data the importance of specific intramolecular interactions for the stability of compact gas-phase protein structure can be inferred. Using this approach, and aided by molecular dynamics simulations, an important stabilising interaction between K6 and H68 in the protein ubiquitin was identified, as was a contact between the N-terminus and E22 in a ubiquitin binding protein UBA2.     

Protein Oligomer Engineering: A New Frontier for Studying Protein Structure,Function, and Toxicity

Prevalent in nature, protein oligomers play critical roles both physiologically and pathologically. The multimeric nature and conformational transiency of protein oligomers greatly complicate a more detailed glimpse into the molecular structure as well as function. In this minireview, the oligomers are classified and described on the basis of biological function, toxicity, and application. We also define the bottlenecks in recent oligomer studies and further review numerous frontier methods for engineering protein oligomers. Progress is being made on many fronts for a wide variety of applications, and protein grafting is highlighted as a promising and robust method for oligomer engineering. These advances collectively allow the engineering and design of stabilized oligomers that bring us one step closer to understanding their biological functions, toxicity, and a wide range of applications.     

Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein

The inhibition of the G protein‐coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41‐residue and three‐disulfide cross‐bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N^α‐deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca²⁺ ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production.     

Cell-Penetrating Peptide–Peptide Nucleic Acid Conjugates as a Tool for Protein Functional Elucidation in the Native Bacterium

Approximately 30% or more of the total proteins annotated from sequenced bacteria genomes are annotated as hypothetical or uncharacterized proteins. However, elucidation on the function of these proteins is hindered by the lack of simple and rapid screening methods, particularly with novel or hard-to-transform bacteria. In this report, we employed cell-penetrating peptide (CPP) –peptide nucleotide acid (PNA) conjugates to elucidate the function of such uncharacterized proteins in vivo within the native bacterium. Paenibacillus, a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed cgiA and cgiB, were identified from the draft genome of Paenibacillus sp. strain YYML68, and CPP–PNA probes targeting the mRNA of the acyl carrier protein gene, acpP, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP–PNA targeting the mRNA of the acpP gene, we successfully observed growth inhibition of strain YYML68 in a concentration-dependent manner. Similarly, both the function of the candidate ι-carrageenases were also inhibited using our CPP–PNA probes allowing for the confirmation and characterization of these hypothetical proteins. In summary, we believe that CPP–PNA conjugates can serve as a simple and efficient alternative approach to characterize proteins in the native bacterium.     

用偶氮胂M分光光度法测定蛋白质

研究了蛋白质与偶氮胂 M的显色反应。在含 0 .0 5 0 % OP的 p H2 .5的缓冲溶液中 ,偶氮胂 M与蛋白质形成兰色复合物 ,λmax为 60 5 nm,ε为 4.5× 1 0 5L· mol- 1· cm- 1,蛋白质含量在 3.3～ 2 5 4 mg/L范围内 ,服从比耳定律。所提出的方法可直接用于血清、花生等生物样品中蛋白质含量测定     

Fluorogenic Photo-Crosslinking of Glycan-Binding Protein Recognition Using a Fluorinated Azido-Coumarin Fucoside

Glycan recognition by glycan-binding proteins is central to the biology of all living organisms. The efficient capture and characterization of relatively weak non-covalent interactions remains an important challenge in various fields of research. Photoaffinity labeling strategies can create covalent bonds between interacting partners, and photoactive scaffolds such as benzophenone, diazirines and aryl azides have proved widely useful. Since their first introduction, relatively few improvements have been advanced and products of photoaffinity labeling remain difficult to detect. We report a fluorinated azido-coumarin scaffold which enables photolabeling under fast and mild activation, and which can leave a fluorescent tag on crosslinked species. Coupling this scaffold to an α-fucoside, we demonstrate fluorogenic photolabeling of glycan-protein interactions over a wide range of affinities. We expect this strategy to be broadly applicable to other chromophores and we envision that such “fluoro-crosslinkers” could become important tools for the traceable capture of non-covalent binding events.     

Electrochemical Single-Cell Protein Therapeutics Using a Double-Barrel Nanopipette

Single-cell protein therapeutics is expected to promote our in-depth understanding of how a specific protein with a therapeutic dosage treats the cell without population averaging. However, it has not yet been tackled by current single-cell nanotools. We address this challenge by the use of a double-barrel nanopipette, in which one lumen was used for electroosmotic cytosolic protein delivery and the other was customized for ionic evaluation of the consequence. Upon injection of protein DJ-1 through the delivery lumen, upregulation of the antioxidant protein could protect neural PC-12 cells against oxidative stress from phorbol myristate acetate exposure, as deduced by targeting of the cytosolic hydrogen peroxide by the detecting lumen. The nanotool developed in this study for single-cell protein therapeutics provides a perspective for future single-cell therapeutics involving different therapeutic modalities, such as peptides, enzymes and nucleic acids.     

基于银增强金标记物的阳极溶出伏安免疫分析用于补体第三成分的测定

分子印迹是制备对特定分子具有专一性结合能力的聚合物的技术,所制备的聚合物被称为分子印迹聚合物（Molecularly imprinted polymers,MIPs）,此类聚合物在分离提纯、模拟酶和传感器等方面均显示出广阔的应用前景,迄今,小分子化合物的印迹技术已经十分成熟。     

Thermodynamic Insight into Protein Aggregation Using a Kinetic Ising Model

In this work, we present a kinetic analysis for protein aggregation using the kinetic Ising model, which serves as a new application of a previously proposed model [Liang et al., J. Chin. Chem. Soc.­ 2003 , 50, 335]. Considering protein as a single spin unit, we map two states of a unit to the aggregation‐prone (AP) and the fibril (F) states. This work shows that the model can successfully capture the nucleation‐growth features of protein aggregation from experiments, which offers thermodynamic interpretations of aggregation properties, such as lag‐time and fibril stability.     

Dip-pen刻蚀技术直接制造蛋白质纳米阵列

美国西北大学 Mirkin等 [1～ 3] 发明的 Dip- pen( DPN,译为蘸水笔技术 )纳米刻蚀技术是以 SPM的针尖为笔 ,通过超分子相互作用使被书写的分子或纳米材料粘在针尖上 ,以某种材料为基底 ,通过合理的超分子相互作用的设计将针尖上的分子或纳米材料书写到基底上 ,从而实现纳米刻蚀和纳米制造的目的 .很显然 ,这种技术对纳米器件、纳米传感器、高密度存储以及生物芯片的制造具有重要意义[4～ 7] .近年来 ,Mirkin研究组和其他几个研究集体利用这种技术成功地制造了有机分子纳米图形与阵列 [8] ;无机氧化物 [9]、金属纳米粒子 [10 ,11]、高分…     

Determination of Trace Amounts of Protein Using Bathocuproinedisulphonic Acid

ＤｅｔｅｒｍｉｎａｔｉｏｎｏｆＴｒａｃｅＡｍｏｕｎｔｓｏｆＰｒｏｔｅｉｎＵｓｉｎｇＢａｔｈｏｃｕｐｒｏｉｎｅｄｉｓｕｌｐｈｏｎｉｃＡｃｉｄＬＩＵＺｈａｏ－ｌａｎ;ＴＩＥＪｉａｎ－ｋｅ;ＺＨＥＮＧＷｅｉ－ｑｉｎｇａｎｄＣＩＹｕｎ－ｘｉａｎｇ（Ｄｅｐａｒ...     

One‐Pot/Sequential Native Chemical Ligation Using N‐Sulfanylethylanilide Peptide

N‐Sulfanylethylanilide (SEAlide) peptides were developed with the aim of achieving facile synthesis of peptide thioesters by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (Fmoc SPPS). Initially, SEAlide peptides were found to be converted to the corresponding peptide thioesters under acidic conditions. However, the SEAlide moiety was proved to function as a thioester in the presence of phosphate salts and to participate in native chemical ligation (NCL) with N‐terminal cysteinyl peptides, and this has served as a powerful protein synthesis methodology. The reactivity of a SEAlide peptide (anilide vs. thioester) can be easily tuned with or without the use of phosphate salts. This interesting property of SEAlide peptides allows sequential three‐fragment or unprecedented four‐fragment ligation for efficient one‐pot peptide/protein synthesis. Furthermore, dual‐kinetically controlled ligation, which enables three peptide fragments simultaneously present in the reaction to be ligated in the correct order, was first achieved using a SEAlide peptide. Beyond our initial expectations, SEAlide peptides have served in protein chemistry fields as very useful crypto‐peptide thioesters. DOI 10.1002/tcr.201200007     

Recognition of Biotin－functionalized Liposomes

Functionalized liposomes were prepared by mixing the biotin in the lipid vesicle suspensions. The experiments through immersing streptavidin deposited mica into the biotin modified liposome solution testify the specifically biological binding interaction and extend the function of liposomes as a biosensor or drug carrier.     

蛋白质快速检测仪测定乳及乳制品中蛋白质

采用研制的蛋白质快速检测仪,系统地考察了温度、时间和干扰物质等因素对蛋白质测定的影响及检测仪的重复性,并将检测仪应用于新鲜乳、纯牛奶、牛奶饮料(核桃、燕麦、红枣)、牛初乳、奶粉、豆奶粉、豆浆粉和鸡蛋等样品中蛋白质的定量测定.实验结果表明,在17～40℃条件下,蛋白质试剂与蛋白质在1 min内即可完成反应,整个蛋白质含量...