Synthesis and conformational studies of peptides fromE. coli pilus proteins recognized by the chaperone PapD

Summary Adhesive pili in uropathogenicE. coli are composed of a few different types of proteins which are assembled into the pilus by the chaperone PapD. Peptides from the C-terminus of these pilus proteins have been prepared by Fmoc solid-phase synthesis. Use of a polyethyleneglycol polystyrene resin was found to be essential for successful synthesis. The conformational propensities of the peptides were analyzed by CD and¹H NMR spectroscopy, with special focus on PapG296-314 from the pilus adhesin which has previously showed the tightest binding to PapD. PapG296-314 was found to be flexible in different solutions, but with a significant propensity to adopt a -conformation. Interestingly the peptide is bound as a -strand in a crystalline complex with PapD. The peptides from three other pilus proteins could only be investigated in trifluoroethanol wher they displayed considerable -helicity in contrast to PapG296-314. These results suggest that conformational factors provide part of the explanation for the differential binding of pilus-related peptides to PapD.Abbreviations CD circular dichroism - CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate - COSY two-dimensional correlated spectroscopy - DMF dimethylformamide - DMSO dimethylsulfoxide - FAB-MS fast-atom-bombardment mass spectroscopy - Fmoc 9-fluorenylmethoxycarbonyl - Gal galactose - HOBt 1-hydroxybenzotriazole - MeCN acetonitrile - MSNT 1-(2-mesitylenesulfonyl)-3-nitro-1,2,4-triazole - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - PEG-PS resin polyethyleneglycol polystyrene resin - ROESY two-dimensional rotating frame nuclear Overhauser enhancement spectroscopy - SDS sodium dodecylsulfate - TFA trifluoroacetic acid - TFE 2,2,2-trifluoroethanol - TOCSY two-dimensional total correlation spectroscopy These authors have made equal contributions to the work presented.correspondence should be addressed.     

Synthesis, conformational studies and biological activities of VIP and related fragments

VIP and related fragments were prepared by the solid-phase method. The peptides were assembled on a benzhydrylamine resin and couplings of the Boc-amino acids were carried out by the symmetrical anhydride method. Cleavage was achieved by treatment with liquid HF and purification was accomplished by successive steps of cation exchange, partition and semi-preparative high pressure liquid chromatography. The biological activities were evaluated in vitro in the rabbit perfused heart and in vivo on the rat blood pressure. Structural studies were performed by high resolution (400 MHz) 1H-NMR spectroscopy and circular dichroism. The results show that among all the fragments tested, only VIP2-28 retains significant biological activity. The fragments 1-14 and 15-28, which are devoid of activity, were found to be inactive as antagonists. VIP and some of the fragments tend to adopt the helical structure, as demonstrated by spectroscopic techniques.     

Total Stepwise Solid-Phase Peptide-Oligonucleotide Conjugate Synthesis on Macroporous Polystyrene

Abstract

An efficient total stepwise solid-phase synthesis of oligonucleotide-peptide conjugates on a macroporous polystyrene is described. Extending our homoserine linker approach, we prepared a range of fluorescein-labelled conjugates containing one of two different peptides together with oligonucleotides containing 2′-deoxynucleoside or 2′-O-methylribonucleoside phosphodiesters, or gapmers containing 2′-deoxyphosphorothioate sequences flanked by 2′-O-methyl wings.     

用混合氨基酸接肽的方法合成OX型肽亚库

肽库合成是药物研究组合化学策略的重要技术之一。建立合成OX型肽亚库的固相合成方法 ,接肽反应采用等摩尔的Fmoc氨基酸混合物和DIC HOBt缩合方法 ,以高浓度的缩合剂和不断缩减溶剂的策略促进偶联反应进行完全。产物的氨基酸组成分析结果显示 ,所使用的常见氨基酸都能以相近的摩尔比例接肽至X位置。推测经多次接肽后最终形成的肽亚库中 ,含低活力氨基酸较多的肽其浓度虽然会较低一些 ,但影响不会太大 ,且本合成方法成本相对较低 ,故可为抗原表位分析、多肽药物筛选及构效关系分析提供一种有用的工具。     

A family of cell-adhering peptides homologous to fibrinogen C-termini

A family of cell-adhesive peptides homologous to sequences on different chains of fibrinogen was investigated. These homologous peptides, termed Haptides, include the peptides Cβ, preCγ, and CαE, corresponding to sequences on the C-termini of fibrinogen chains β, γ, and αE, respectively. Haptides do not affect cell survival and rate of proliferation of the normal cell types tested. The use of new sensitive assays of cell adhesion clearly demonstrated the ability of Haptides, bound to inert matrices, to mediate attachment of different matrix-dependent cell types including normal fibroblasts, endothelial, and smooth muscle cells. Here we present new active Haptides bearing homologous sequences derived from the C-termini of other proteins, such as angiopoietin 1&2, tenascins C&X, and microfibril-associated glycoprotein-4. The cell adhesion properties of all the Haptides were found to be associated mainly with their 11 N-terminal residues. Mutated preCγ peptides revealed that positively charged residues account for their attachment effect. These results suggest a mechanism of direct electrostatic interaction of Haptides with the cell membrane. The extended Haptides family may be applied in modulating adhesion of cells to scaffolds for tissue regeneration and for enhancement of nanoparticulate transfection into cells.     

Identification of bovine CD52-like molecule by monoclonal antibody IVA-543: distribution of CD52-like molecule in the bull genital tract

The bovine maturation-associated sperm membrane antigen CD52-like molecule has been analysed using a mouse anti-sperm monoclonal antibody developed against bull spermatozoa. The antigen recognised by monoclonal antibody IVA-543 was detected on blood mononuclear cells (including lymphocytes and monocytes) and on a minor population of polymorphonuclear leukocytes. The bovine CD52-like molecule is secreted by the epididymal epithelium and then it is inserted into the sperm membrane during the epididymal transport in the distal part of epididymis. The CD52-like molecule was absent from spermatozoa derived from testes, and the highest proportion of IVA-543-reactive sperm was observed in the cauda epididymis (91.6%).This study has shown that the new molecule identified on bovine cells has properties analogous to those previously described for CD52 molecules in man, mouse, rat, monkey, and dog.     

部分氧化的铁钼蛋白的圆二色散谱和电子顺磁共振谱的研究

棕色固氮菌固氮酶FeMo蛋白与过量（5－6个当量）的酸性靛蓝保温30－60分钟后,蛋白中的P－金属原子族全部氧化,然而蛋白中的FeMoCo全都处于还原状态。Na2S2O4使这种部分氧化的FeMo蛋白中的P-ciuster重新不,甲基紫精可加快这种还原,而亚甲蓝等氧化剂则使这种蛋白中的FeMoCo受到氧化,对这种部分氧化的FeMo蛋白分别进行CD还原滴定和测定氧化过程中的EPR／ABS的变化已经得到p-Cluster和FeMoCo的氧化还原当量数目。     

Studies on the Circular Dichroism (CD) and Electron Paramaguetic Resonance(EPR) Spe-ctroscopy of the Partially Oxidized FeMo Protein from Azotobacter vinelandii Nitrogenase

After nitrogenase FeMo protein from A. vinelandii was incubated with a large excess (5–6 equivalents) of indigo carmine for 30–60 min., all of P-clusters in the partially oxidized FeMo protein were oxidized, but all of FeMoCo in the protein were still in the reductive state. The oxidized P-clusters in the protein were able to be completely reduced by Na2S2O4(DT) and the reduction was accelerated by methyl viologen (MV). And all of FeMoCo in the protein were firstly oxidized by some oxidants such as methylene blue. The numbers of the redox equivalent of P-cluster and FeMoCo have been obtained by the CD reductive titration of and EPR/ ABS time course on the oxidation of the partially oxidizeA FeMo protein, respectivelly.     

猪繁殖与呼吸综合征病毒N蛋白的原核表达、纯化与结构分析

将猪繁殖与呼吸综合征病毒 (PRRSV)BJ 4毒株N基因克隆至原核表达载体pET2 8a中 ,得到重组表达载体pET2 8 N ,转化EscherichiacoliBL2 1(DE3)细胞 ,获得可溶性表达 ,表达量占菌体蛋白的 2 8%。经ProbandNi2 亲和层析获得重组蛋白P2 8 N ,圆二色谱 (CD)测定结果表明 ,P2 8 N重组蛋白螺旋占 2 6 1% ,折叠占 2 3 7% ,转角 19 8% ,卷曲占 30 3%。并进一步绘制出PRRSVN蛋白的二级结构图     

Conformational studies of alanine-rich peptide using CD and FTIR spectroscopy.

The circular dichroism (CD) and Fourier transform infrared (FTIR) methods were applied to the conformational studies of alanine-rich peptide Ac-K-[A]11-KGGY-NH2 (where K is lysine, A is alanine, G is glycine and Y is tyrozyne) in water, methanol (MeOH) and trifluoroethanol (TFE). The analysis of CD-spectra of the peptide in water at different concentrations revealed that the secondary structure content depends on the peptide concentration and pH of the solution. The increase of the peptide concentration causes a decrease of alpha-helix content and, simultaneously, an increase of beta-sheet structure, while the unordered structure is the predominant one. Additional elements are discovered in MeOH and TFE but alpha-helix and beta-turns predominate. Moreover, in these solutions the percentage content of the secondary structure does not depend on the temperature. FTIR measurements, carried out at higher peptide concentration (about one order of magnitude) than these CD measurements mentioned above, revealed that in water solution the solid state beta-sheet, and aggregated structures, dominate. However, in TFE the most abundant are alpha-helix and beta-turns structures. The thioflavine T assay showed the tendency of the studied peptide for aggregate.     

Effect of surfactants on the thermal, conformational and rheological properties of collagen

Collagen is an important biomaterial and its interaction with surfactant is important in light of its use in various cosmetics and dermatological applications. Presently, the effect of surfactants on the physico-chemical properties of collagen has been studied. The thermal stability of collagen is reduced by sodium dodecyl sulfate and hexadecyltrimethylammmonium bromide, whereas Triton X-100 does not. The viscosity of collagen is influenced greatly depending on the surfactant concentrations. The secondary structure of collagen shows changes only in the molar ellipticity. The role of charge and concentration of surfactants in influencing the various physico-chemical properties of collagen has been elucidated.     

Interaction of trifluoperazine with porcine calmodulin F NMR and induced CD spectral studies

We have used ¹⁹F NMR and CD spectra to study interactions of TFP with CaM under various conditions where no aggregation of TFP occurs. It was found that TFP is bound to CaM in the absence of Ca²⁺ with K_a >4 × 10⁴ M in terms of ¹⁹F NMR of TFP and that KCl markedly influences the interaction of TFP with CaM. It was also found that TFP causes quite strong induced CD bands corresponding with the absorption bands of the aromatic ring of TFP when TFP is bound to Ca²⁺ -CaM. The induction of the CD bands was time-dependent and was composed of fast and slow phases.     

NMR, biophysical, and biochemical studies reveal the minimal Calmodulin binding domain of the HIV-1 matrix protein

Subcellular distribution of Calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM has been shown to interact and co-localize with the HIV-1 Gag protein in infected cells. However, the precise molecular mechanism of this interaction is not known. Binding of Gag to CaM is dependent on calcium and is mediated by the N-terminal-myristoylated matrix (myr(+)MA) domain. We have recently shown that CaM binding induces a conformational change in the MA protein, triggering exposure of the myristate group. To unravel the molecular mechanism of CaM-MA interaction and to identify the minimal CaM binding domain of MA, we devised multiple approaches utilizing NMR, biochemical, and biophysical methods. Short peptides derived from the MA protein have been examined. Our data revealed that whereas peptides spanning residues 11-28 (MA-(11-28)) and 31-46 (MA-(31-46)) appear to bind preferentially to the C-terminal lobe of CaM, a peptide comprising residues 11-46 (MA-(11-46)) appears to engage both domains of CaM. Limited proteolysis data conducted on the MA-CaM complex yielded a MA peptide (residues 8-43) that is protected by CaM and resistant to proteolysis. MA-(8-43) binds to CaM with a very high affinity (dissociation constant = 25 nm) and in a manner that is similar to that observed for the full-length MA protein. The present findings provide new insights on how MA interacts with CaM that may ultimately help in identification of the functional role of CaM-Gag interactions in the HIV replication cycle.     

Expression of CD52 mRNA in the rat embryo

CD52 is a leukocyte differentiation antigen first discovered in humans as expressed on the surface of lymphocytes, monocytes and eosinophils. The human CD52 is found on chromosome 1, and two alleles are both known to be reasonably common. A closely homologous gene has been identified in the cynomologous monkey and related genes have been found in mouse, rat and dog. The role of CD52 in lymphocyte is still unclear but the anti-CD52 antibodies named CAMPATH-1 antibodies are largely used for therapy where depletion of lymphocytes is required. In the past expression of the antigen on progenitors of leukocytes in bone marrow had been excluded, but recent work indicates CD52 is highly expressed on cells with colony-forming and NOD/SCID (non-obese diabetic-severe combined immunodeficiency)-engrafting capacities, both at the mRNA and membrane protein level. We have investigated CD52 expression during development in rat embryos by in situ hybridization. We report here that the antigen is highly expressed in the liver that is the major organ where multipotent hematopietic stem cells differentiate but also in the splancnopleuric mesoderm, at early stages of embryo differentiation, where hematopietic stem cells are suggested to arise. CD52⁺ cells were found in areas active in vasculogenesis at early embryo stages and in the walls of the vessels in the liver at mid gestation. CD52⁺ cells were also found to emerge among c-Kit positive cells.     

Synthesis and CD studies of an 88-residue peptide containing the main receptor binding site of HTLV-I SU-glycoprotein

Essential HTLV-I biological functions, like host-cell receptor recognition, depend on the structural motives on the surface glycoprotein gp46. We defined a peptide of 88 amino acids [Arg¹⁴⁷-Leu²³⁴] corresponding to the central part of the protein sequence, where major neutralizing epitopes are localized. After evaluating the feasibility of its chemical synthesis, the chosen sequence was realized using the stepwise solid-phase methodology. Multiple chromatographic purification steps were required to obtain a sample suitable for structural analysis. Correct folding was supported by strong binding of monooclonal antibodies, recognizing known exposed immunodominant regions. Circular dichroism studies confirmed a non-random conformation of at least 70–80% of the synthetic peptide. Investigation of the 3D-structure of the synthetic peptide will provide useful information for future vaccine and drug-design strategies. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci.3: 347–353 No. of Figures: 5. No. of Tables: 0. No. of References: 23     

Studies on the Circular Dichroism (CD) Spectra of Iron-Molyb-denum Protein from Azotobacter vinelandii Nitrogenase

Since the unsymmetry of metal cluster(P-cluster), which was under different redox states, in FeMo protein of A. vinelandii nitrogenase could be observed by circular dichroism, the numbers of the redox equivalent for the P-cluster was obtained by CD titration of DT-stripped FeMo protein with oxidant, i.e. plotting △δ450nm against the number of equivalent of oxidant added. After exposure to air, at the beginning P-cluster was reversibly oxidized, then followed by irreversible oxygen-damage. Dithiothreitol (DTT) was able to increase the C2H2-reduction activity of FeMo protein, which was not severely damaged by O2, or by other stronger oxidants, but could not change their CD spectra. It seems that the reactivation may be not due to the restoration of the P-cluster to its reducing state.     

Regioselective Disulfide Solid Phase Synthesis, Chemical Characterization and In Vitro Receptor Binding Activity of Equine Relaxin

In the equine industry, pregnancy loss during the third trimester constitutes a large percentage of fetal and neonatal mortality and represents a major financial loss and time investment for the breeder. Early identification of placental insufficiency would, in some cases, make it possible to sustain the pregnancy through medical intervention. Recent work suggests that relaxin is a valuable clinical tool for diagnosing placental insufficiency and monitoring treatment efficacy in mares. Relaxin is a polypeptide member of the insulin superfamily that consists of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. It is typically produced in the ovary during pregnancy and has primary roles in maintaining mammalian pregnancy and facilitating the delivery of the young via remodelling of the reproductive tract. The placenta is the primary source of relaxin in the mare during pregnancy. Its primary structure has been determined and shown to be the smallest of the known mammalian relaxins. It consists of a 20 residue A-chain and a 28-residue B-chain. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken using regioselective disulfide formation methods. The synthetic equine relaxin showed typical α-helical structure under physiological conditions. The peptide was found to bind to the relaxin receptor, LGR7, in vitro, and its binding affinity was found to be higher than that of the “gold standard”, porcine relaxin, and similar to that of the human relaxin-2 (H2 relaxin).     

一组人工合成抗菌肽的研究

Cecropin A1是一种从惜古比天蚕(Hyalophora cecropia)中提取的由37个氨基酸组成的一种α-螺旋抗菌肽,其杀菌活性较弱。本文采用了cecropin A1的N端1-8序列KWKLFKKI,另加一段标准的α-螺旋结构序列,然后用一个铰链结构GIG相连,合成了15条抗菌肽。经过试验证明部分含有核心标准螺旋结构的序列,对革兰氏阳性菌和阴性菌的最小抑菌浓度仅是原有cecropin A1抗菌肽的1/100左右。该类抗菌肽有希望进一步开发为新的抗感染药物。该类抗菌肽已经申请专利,专利号为PCT/CN 03/00522。     

Circular dichroism study on the secondary structural change induced by complex formation of a peptide derived from a CD4 binding site of HIV-1 envelope glycoprotein gp120 and a peptide from the N-terminal domain of CD4

Summary Circular dichroism spectroscopy was used to study the conformational change of a peptide containing a CD4 binding region of HIV-1 envelope glycoprotein gp120 complexed with a CD4 fragment. In free solution the gp120 peptide exists primarily as -sheet and random coil. Upon association with the peptide, encompassing a critical gp120 binding site on the extracellular domain 1 of CD4, the -helical content of the complex relative to that of the two component peptides increases significantly, at the expense of random coil and turn. An increase in the helix structure for the gp120 peptide, but not the CD4 peptide, was observed in 30% trifluoroethanol (TFE)/H₂O (v:v) solution. The conformational change in the gp120 C4 peptide when complexing with CD4 is proposed as part of the process that facilitates the membrane fusion between the virion and its target cell.Abbreviations CD circular dichroism - HIV human immunodeficiency virus - AIDS acquired immunodeficiency syndrome - Fmoc 9-fluorenylmethoxycarbonyl - mAb monoclonal antibody - gp glycoprotein - TFE trifluoroethanol - HPLC high-performance liquid chromatography     

Synthesis, and structural and biological studies of efrapeptin C analogues

A series of analogues of efrapeptin C (1), with variations in the central tripeptide epitope (positions 6-8), were prepared by a combination of solid- and solution-phase peptide syntheses. The conformations of the modified compounds 2-6 were investigated by circular-dichroism (CD) spectroscopy to differentiate between 3(10)- and alpha-helical secondary structures. The inhibitory activities of the new compounds towards F(1)-ATPase from E. coli were determined. The modified congeners 3-5 were less active by one order of magnitude compared to 1 (K(i) 10 microM), and 6 was completely inactive. Our experiments demonstrate that the flexible, central tripeptide epitope, comprising positions 6-8 in 1, is crucial for molecular recognition, even slight sequence modifications being hardly tolerated.