A simple approach to well-defined sugar-coated surfaces for interaction studies

Protein-carbohydrate interactions play a crucial role in many relevant biological processes, and the development of simple and reliable tools for their study is a well-recognized need. Surface-based methods are particularly attractive because they i) can effectively mimic cell-surface recognition events, ii) allow the identification of low-affinity binders, iii) are easily adaptable to high-throughput screening, and iv) require minimal sample amounts. We describe here the design and synthesis of a peptide module that efficiently captures glycans through its reducing end, by oxime ligation. Immobilization to carboxyl-functionalized supports was thereby made possible. Chemically well-defined surfaces coated with selected glycan targets were generated by this approach for surface plasmon resonance (SPR) studies. The usefulness of the method was demonstrated in the analysis of interactions that covered a five-orders-of-magnitude affinity range; namely, the strong binding (KA approximately 10(9) M(-1)) of a well-known lectin (wheat germ agglutinin) to chitopentose (GlcNAc5), and that of the same sugar with a weak binder (KA approximately 10(4) M(-1)), HEV32--the smallest hevein domain described.     

The role of asparagine-32 in forming the receptor-binding epitope of human epidermal growth factor

The highly conserved asparagine residue at position 32 (Asn32)in the 'hinge' region of epidermal growth factor (EGF) separatesthe N- and C-terminal structural motifs of the EGF moleculeand is therefore an appropriate target for structure-functionstudies. Analogs of human EGF (hEGF) were generated in whichAsn32 was substituted with aspartate, glycine, isoleucine, lysine,proUne and tryptophan. The relative affinity of the EGF receptorfor mutant hEGF analogs was determined by radioreceptor competitionassay. A wide range of receptor affinities was observed dependingon the amino acid substitution. N32K and N32W hEGF analogs hadrelatively high receptor affinity, while the N32G and N32D analogsshowed decreased affinity, 35% and 25% respectively, relativeto wild type hEGF. However, no binding of the N32P analog wasdetected by radioreceptor competition assay. The N32P mutantdisplayed an NMR spectrum significantly different from thatof native wild type hEGF, indicating gross structural perturbation.In contrast, the N32K and N32D analogs exhibited spectra similarto that of native wild type hEGF. Genetically combining theN32D hEGF with an hEGF species having either the mutation L26Ghi the N-terminal region or L47A in the C-terminal region, generateddouble-mutant hEGF species whkh had relative affinities essentiallyequal to the product of the relative affinities of the parenthEGF mutants, indicating functionally independent changes inUgand-receptor interaction. These studies indicate the requirementfor H-bond donor functionality in the side chain of residuenumber 32 in forming a fully competent receptor-binding epitope.     

N,N,N',N'-四(3-氨基丙基)乙二胺合成和表征

利用乙二胺和丙烯腈的亲核加成反应合成了一种新型有机多腈N,N,N’,N’-四（2-氰基乙基）乙二胺。在Raney Ni存在下对N,N,N’N’-四（2-氰基乙基）乙二胺进行催化加氢,得到N,N,N’N’-四（3-氨基丙基）乙二胺。并用红外、核磁、质谱等对N,N,N’N’-四（3-氨基丙基）乙二胺进行了表征,结果表明所合成的化合物即为标题化合物。     

两种大环氨基膦酸及其La^3＋,Gd^3＋络合物的合成

在浓酸存在下,１,４,７,１０－四氮杂环十二烷和１,４,８,１１－四氮杂环十四烷分别与亚磷酸,甲醛发生Ｍａｎｎｉｃｈ型反应,得到相应的Ｎ,Ｎ”,Ｎ″,Ｎ″－四甲膦酸－１,４,７,１０－四氮杂环十二烷（ＤＯＴＰ）和Ｎ,Ｎ′,Ｎ″,Ｎ″－四甲膦酸－１,４,８,１１－四氮杂环十四烷（ＴＥＴＰ）,产物纯度较高,在不同的ｐＨ值条件下测定了产物的ＨＮＭＲ,考察了其化学位移的变化,在此基础上,采用改进的大环配体     

The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy

The stability and unfolding of an immunoglobulin (Ig) G bindingprotein based upon the B domain of protein A (SpAB) from Staphylococcusaureus were studied by substituting tryptophan residues at strategiclocations within each of the three a-helical regions (al-a3)of the domain. The role of the C-terminal helix, a3, was investigatedby generating two protein constructs, one corresponding to thecomplete SpAB, the other lacking a part of ct3; the Trp substitutionswere made in both one-and two-domain versions of each of theseconstructs. The fluorescence properties of each of the single-tryptophanmutants were studied in the native state and as a function ofguanidine-HCl-mediated unfolding, and their IgG binding activitieswere determined by a competitive enzyme-linked immunosorbentassay. The free energies of folding and of binding to IgG foreach mutant were compared with those for the native domains.The effect of each substitution upon the overall structure andupon the IgG binding interface was modelled by molecular graphicsand energy minimization. These studies indicate that (i) 3 contributesto the overall stability of the domain and to the formationof the IgG binding site in l and 2, and (ii) al unfolds first,followed by 2 and 3 together.     

Structure and Energetics of Ligand–Fluorine Interactions with Galectin-3 Backbone and Side-Chain Amides: Insight into Solvation Effects and Multipolar Interactions

Multipolar fluorine–amide interactions with backbone and side-chain amides have been described as important for protein–ligand interactions and have been used to improve the potency of synthetic inhibitors. In this study, fluorine interactions within a well-defined binding pocket on galectin-3 were investigated systematically using phenyltriazolyl-thiogalactosides fluorinated singly or multiply at various positions on the phenyl ring. X-ray structures of the C-terminal domain of galectin-3 in complex with eight of these ligands revealed potential orthogonal fluorine–amide interactions with backbone amides and one with a side-chain amide. The two interactions involving main-chain amides seem to have a strong influence on affinity as determined by fluorescence anisotropy. In contrast, the interaction with the side-chain amide did not influence affinity. Quantum mechanics calculations were used to analyze the relative contributions of these interactions to the binding energies. No clear correlation could be found between the relative energies of the fluorine–main-chain amide interactions and the overall binding energy. Instead, dispersion and desolvation effects play a larger role. The results confirm that the contribution of fluorine–amide interactions to protein–ligand interactions cannot simply be predicted, on geometrical considerations alone, but require careful consideration of the energetic components.     

含磷的金属螯合剂──N，N，N＇，N＇－烃基二氨基四亚甲基膦酸的合成研究（Ⅲ）

ＥＤＴＭＰ［Ｎ，Ｎ，Ｎ＇，Ｎ＇－乙二氨基四亚甲基膦酸］和ＨＤＴＰ［Ｎ，Ｎ，Ｎ＇，Ｎ＇－ｌ，６－己二氨基四亚甲基膦酸］是一类重要的金属螯合剂。以甲醛、三氯化磷、乙二胺和己二胺为原料合成了ＥＤＴＭＰ和ＨＤＴＰ，经柱色谱分离，产率在５６％以上，含量大于９８％。     

氮四取代苯二胺新型除氧剂的研究

介绍了氮四取代苯二胺类新型除氧剂的除氧性能，选取其中的N，N，N'，N'－四甲基对苯二胺为主要研究对象，与N2H4和丙酮肟的除氧性能进行了对比，研究了其缓蚀性能。试验结果表明，氮四取代苯二胺是一种性能优良的新型除氧剂。     

新型聚季铵盐的合成、表征及其性能测试

以二氯乙醚和N,N,N’,N’-四甲基乙二胺为原料,通过共聚合反应制备出新型的水溶性聚季铵盐——聚[(氧亚乙基-二甲基亚氨基-亚乙基-二甲基亚氨基-亚乙基)二氯化物]。研究表明,最佳合成工艺条件为:四甲基乙二胺和二氯乙醚的摩尔配比为1.05∶1,反应温度控制在70℃,反应时间为6 h,滴加速度为30滴/min。采用红外、核磁共振对产物进行结构表征;测定了聚合物的热稳定性,特征粘数等;初步测试了该聚季铵盐的杀菌性能;杀菌时间9 h,药剂投加量为80 mg/L时杀菌效果最好,杀菌率达到98%;产物最大特征粘数为15.231 3 mL/g。     

硅粉凝胶注模成型的研究

以硅粉为原料,通过对硅粉悬浮液的Zeta电位、粘度、分散性的测试,制备了固相体积比为50vol%硅粉的稳定悬浮液;通过调节引发剂过硫酸铵、催化剂N,N,N',N'-四甲基乙二胺(TEMED),制备了凝胶时间为1小时,干燥强度为7MPa的硅材料坯体。     

孪尾疏水单体N,N-二正十二烷基丙烯酰胺的合成与表征

用十二胺和1 溴代十二烷合成了二正十二胺,产率73%。以NaOH作HCl的吸收剂,利用二正十二胺与丙烯酰氯非均相胺解法合成了孪尾疏水单体N,N 二正十二烷基丙烯酰胺,投料比为n(DiC12)∶n(AC)(摩尔比)=1∶1.25时,在0～10℃下,反应6h,产率可达89.23%。用FTIR、1HNMR对二正十二胺、N,N 二正十二烷基丙烯酰胺进行了表征,还用元素分析法对N,N 二正十二烷基丙烯酰胺进行了组分分析。     

Antagonism, non-native interactions and non-two-state folding in S6 revealed by double-mutant cycle analysis

When folding to the native state N in the presence of salt, the apparent two-state folder S6 transiently forms a transient off-pathway state C with substantial secondary and tertiary structure. Fifteen double mutant cycles were analysed to compare side-chain interaction energies DeltaDeltaG(int) in C, N and TS (the transition state between N and the denatured state). The kinetic signatures of these destabilizing mutants suggest folding scenarios involving unfolding intermediates and even alternative unfolding pathways. However, restricting the kinetic data to linear parts of the chevron plot allows reliable extrapolation to zero molar denaturant of rate constants of folding, unfolding and misfolding. Side-chain interactions appear to contribute to the stability of C, but in a substantially non-native environment, as shown by changes in the sign of DeltaDeltaG(int) between C and N. Remarkably, there appear to be significant (0.7-2 kcal/mol) antagonistic interactions between the two residues Leu30 and Leu75 in N and TS, which may be linked to subtle structural changes seen in the crystal structures of the mutants. A small number of overlapping residues are involved in these kinds of antagonistic interactions in N, TS and C, suggesting that repulsive interactions are coded into the protein topology whether the protein folds or misfolds. Destabilizing double mutants indicate that apparent two-state folders can be induced to behave in more complex ways provided that the native state is suitably destabilized.     

SAPO-56分子筛的晶化历程及其催化性能

以N,N,N′,N′-四甲基-1,6-己二胺（TMHD）为模板剂，采用水热法合成SAPO?-56分子筛。用NMR、XRD及SEM等测试手段进行表征，研究了不同晶化时间对SAPO-56分子筛合成的影响，并考察了甲醇转化制低碳烯烃（MTO）的反应性能。     

The synthesis and fluorescence behaviour of new unsymmetrically mono-functionalized carboxy Ge, Ti and Sn phthalocynines

This work reports on the synthesis and fluorescence behaviour of novel unsymmetrically substituted monocarboxy germanium ((OH)₂GeMCPc, 3), titanium (OTiMCPc 4) and tin ((ac)₂SnMCPc, 5) phthalocyanines. The fluorescence quantum yields ranged from 0.09 to 0.14. The fluorescence lifetimes were found to be higher for the complex with higher fluorescence quantum yield value. Higher fluorescence quantum yields and lifetimes were obtained for the (ac)₂SnMCPc complex (5), followed by OTiMCPc complex (4), and the lowest fluorescence quantum yield and lifetime were observed for (OH)₂GeMCPc (3).     

Discovery, structure-activity relationship studies, and crystal structure of nonpeptide inhibitors bound to the Shank3 PDZ domain

Shank is the central scaffolding protein of the postsynaptic density (PSD) protein complex found in cells of the central nervous system. Cellular studies indicate a prominent role of the protein in the organization of the PSD, in the development of neuronal morphology, in neuronal signaling, and in synaptic plasticity, thus linking Shank functions to the molecular basis of learning and memory. Mutations in the Shank gene have been found in several neuronal disorders including mental retardation, typical autism, and Asperger syndrome. Shank is linked to the PSD complex via its PDZ domain that binds to the C‐terminus of guanylate‐kinase‐associated protein (GKAP). Here, small‐molecule inhibitors of Shank3 PDZ domain are developed. A fluorescence polarization assay based on an identified high‐affinity peptide is established, and tetrahydroquinoline carboxylates are identified as inhibitors of this protein–protein interaction. Chemical synthesis via a hetero‐Diels–Alder strategy is employed for hit optimization, and structure–activity relationship studies are performed. Best hits possess K_i values in the 10 μM range, and binding to the PDZ domain is confirmed by ¹H,¹⁵N HSQC NMR experiments. One of the hits crystallizes with the Shank3 PDZ domain. The structure, analyzed at a resolution of 1.85 Å, reveals details of the binding mode. Finally, binding to PDZ domains of PSD‐95, syntrophin, and DVL3 was studied using ¹H,¹⁵N HSQC NMR spectroscopy.     

Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies

RNA helicases represent a family of enzymes that unwind double-stranded(ds) RNA in a nucleoside triphosphate (NTP)-dependent fashionand which are required in all aspects of cellular RNA metabolismand processing. The hepatitis C virus (HCV) non-structural 3(NS3) protein possesses a serine protease activity in the N-terminalone-third, whereas RNA-stimulated NTPase and helicase activitiesreside in the C-terminal portion of the 631 amino acid residuebifunctional enzyme. The HCV NS3 RNA helicase is of key importancein the life cycle of HCV, which makes it a target for the developmentof therapeutics. However, neither the precise mechanism northe substrate structure has been defined for this enzyme. Fornuclear magnetic resonance (NMR)-based drug discovery methodsand for mechanistic studies we engineered, prepared and characterizedvarious truncated constructs of the 451-residue HCV NS3 RNAhelicase. Our goal was to produce smaller fragments of the enzyme,which would be amenable to solution NMR techniques while retainingtheir native NTP and/or nucleic acid binding sites. Solutionconditions were optimized to obtain high-quality heteronuclearNMR spectra of nitrogen-15 isotope-labeled constructs, whichare typical of well-folded monomeric proteins. Moreover, NMRbinding studies and functional data directly support the correctfolding of these fragments.     

Analysis of Conformational Motions and Residue Fluctuations for Escherichia coli Ribose-Binding Protein Revealed with Elastic Network Models

The ribose-binding protein (RBP) is a sugar-binding bacterial periplasmic protein whose function is associated with a large allosteric conformational change from an open to a closed conformation upon binding to ribose. The open (ligand-free) and closed (ligand-bound) forms of RBP have been found. Here we investigate the conformational motions and residue fluctuations of the RBP by analyzing the modes of motion with two coarse-grained elastic network models, the Gaussian Network Model (GNM) and Anisotropic Network Model (ANM). The calculated B-factors in both the calculated models are in good agreement with the experimentally determined B-factors in X-ray crystal structures. The slowest mode analysis by GNM shows that both forms have the same motion hinge axes around residues Ser103, Gln235, Asp264 and the two domains of both structures have similar fluctuation range. The superposition of the first three dominant modes of ANM, consisting of the rotating, bending and twisting motions of the two forms, accounts for large rearrangement of domains from the ligand-free (open) to ligand-bound (closed) conformation and thus constitutes a critical component of the RBP’s functions. By analyzing cross-correlations between residue fluctuation and the difference-distance plot, it is revealed that the conformational change can be described as a rigid rotation of the two domains with respect to each other, whereas the internal structure of the two domains remains largely intact. The results directly indicate that the dominant dynamic characteristics of protein structures can be captured from their static native state using coarse-grained models.     

Solvent-dependent selective fluorescence sensing of Al3 + and Zn2 + using a single Schiff base

The selective assay of Al^3 + and Zn^2 + ions is reported using fluorescence enhancement of a simple Schiff base receptor 1 (1 = 1-((E)-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-ylimino)methyl)naphthalen-2-ol) in aqueous solvents. The Schiff base receptor 1 showed a turn-on fluorescence in the presence of Al^3 + in a mixture of methanol–water, as a result of a restricted CN isomerization mechanism. The ion selectivity of 1 could be switched for Zn^2 + by swapping the solvent from methanol–water to DMF–water mixture. In particular, this chemosensor could clearly distinguish Zn^2 + from Cd^2 +. The binding properties of 1 with the metal ions were investigated by UV–vis, fluorescence, electrospray ionization mass spectroscopy and ¹H NMR titration.     

Spectroscopic properties of an engineered maltose binding protein

The maltose binding protein (MBP) has been site specifically labelled with a nitrobenzoxadiazole (NBD) group following mutation of a serine to a cysteine residue at position 337. The resulting protein shows a large ligand (maltose or beta-cyclodextrin) dependent increase in its steady-state fluorescence intensity. Analysis of the static (intensity and anisotropy) and dynamic (lifetime distributions) fluorescence of the NBD label as well as the tryptophan residues in both ligand-bound and ligand-free states of this molecule reveals complex multi-component decays that are interpreted in terms of a ligand-induced solvent shielding mechanism. In the context of the known crystal structures of the various forms of the maltose-binding protein (MBP), ligand- dependent changes in both the fluorescence parameters as well as the circular dichroism spectra of the NBD group are interpreted by a twisted intramolecular charge-transfer (TICT) mechanism, wherein ligand binding locks the NBD group into a conformation that prevents efficient relaxation of the excited state.