NMR characterization of surface interactions in the cytochrome b5-cytochrome c complex

The complex formed in solution by native and chemically modified cytochrome c with cytochrome b5 has been studied by 1H and 13C nuclear magnetic resonance spectroscopy (NMR). Contrary to predictions of recent theoretical analysis, 1H NMR spectroscopy indicates that there is no major movement of cytochrome c residue Phe82 on binding to cytochrome b5. The greater resolution provided by 13C NMR spectroscopy permits detection of small perturbations in the environments of cytochrome c residues Ile75 and Ile85 on binding with cytochrome b5, a result that is in agreement with earlier model-building experiments. As individual cytochrome c lysyl residues are resolved in the 1H NMR spectrum of N-acetimidylated cytochrome c, the interaction of this modified protein with cytochrome b5 has been studied to evaluate the number of cytochrome c lysyl residues involved in binding to cytochrome b5. The results of this experiment indicate that at least six lysyl residues are involved, two more than predicted by static model building, which indicates that cytochrome c and cytochrome b5 form two or more structurally similar 1:1 complexes in solution.     

一种新型乳清蛋白肽的核磁共振研究

乳清蛋白经碱性蛋白酶酶解的产物,经超滤、凝胶过滤色谱以及反向高效液相色谱分离纯化得到一种纯度较高的多肽。多肽经液质分析,初步确定其氨基酸序列为Asp-Gln-Trp-Leu,为一种新的肽段。本文采用核磁共振波谱法[包括~1H、~(13)C、~1H-~1H COSY(~1H-~1H correlation spectroscopy),HMBC(heteronuclear multiple bond correlation),HMQC(heteronuclear multiple quantum coherence),TOCSY(total correlation spectroscopy),NOESY(nuclear overhauser effect spectroscopy)]对其结构进行了解析,并对核磁信号进行了归属。实验结果显示TOCSY、HMBC和NOESY技术在多肽核磁共振信号的归属中发挥着关键的作用。     

NMR studies of the structure and dynamics of membrane-bound bacteriophage Pf1 coat protein

Filamentous bacteriophage coat protein undergoes a remarkable structural transition during the viral assembly process as it is transferred from the membrane environment of the cell, where it spans the phospholipid bilayer, to the newly extruded virus particles. Nuclear magnetic resonance (NMR) studies show the membrane-bound form of the 46-residue Pf1 coat protein to be surprisingly complex with five distinct regions. The secondary structure consists of a long hydrophobic helix (residues 19 to 42) that spans the bilayer and a short amphipathic helix (residues 6 to 13) parallel to the plane of the bilayer. The NH2-terminus (residues 1 to 5), the COOH-terminus (residues 43 to 46), and residues 14 to 18 connecting the two helices are mobile. By comparing the structure and dynamics of the membrane-bound coat protein with that of the viral form as determined by NMR and neutron diffraction, essential features of assembly process can be identified.     

Studying enzyme mechanism by 13C nuclear magnetic resonance

High-resolution carbon-13 nuclear magnetic resonance (NMR) spectra of enzyme-inhibitor and enzyme-substrate complexes provide detailed structural and stereochemical information on the mechanism of enzyme action. The proteases trypsin and papain are shown to form tetrahedrally coordinated complexes and acyl derivatives with a variety of compounds artificially enriched at the site or sites of interest. These results are compared with the structural information derived from x-ray diffraction. Detailed NMR studies have provided a clearer picture of the ionization state of the residues participating in enzyme-catalyzed processes than other more classical techniques. The dynamics of enzymic catalysis can be observed at sub-zero temperatures by a combination of cryoenzymology and carbon-13 NMR spectroscopy. With these powerful techniques, transient, covalently bound intermediates in enzyme-catalyzed reactions can be detected and their structures rigorously assigned.     

Calcium-induced peptide association to form an intact protein domain: 1H NMR structural evidence

The 70-residue carboxyl-terminal domain of the muscle contractile protein troponin-C contains two helix-loop-helix calcium (Ca)-binding sites that are related to each other by approximate twofold rotational symmetry. Hydrophobic residues from the helices and a short three residue beta sheet at the interface of the two sites act to stabilize the protein domain in the presence of Ca. A synthetic 34-residue peptide representing one of these sites (site III) has been synthesized and studied by H-1 nuclear magnetic resonance (NMR) spectroscopy. In solution this peptide undergoes a Ca-induced conformational change to form the helix-loop-helix Ca-binding motif. Two-dimensional nuclear Overhauser effect spectra have provided evidence for the formation of a beta sheet and interactions between several hydrophobic residues from opposing helices as found in troponin-C. It is proposed that a symmetric two-site dimer similar in tertiary structure to the carboxyl-terminal domain of troponin-C forms from the assembly of two site III peptides in the Ca-bound form.     

Antimicrobial Property of 2-Hydroxypropane-1,2,3-Tricarboxylic Acid Isolated from Citrus microcarpa Extract

This article described antimicrobial property and structure analysis of 2-hydroxypropane-1,2,3-tricarboxylic acid isolated from the crude extract of Citrus microcarpa. Presently, there was no report on compound from C. microcarpa that possessed antimicrobial property against fish pathogenic bacteria. Therefore, in this study, the bioactive principle in C. microcarpa extract was isolated using thin layer chromatography. It＇s structure was elucidated based on nuclear magnetic resonance （NMR） spectroscopic data, such as proton NMR （1HNMR）, correlation spectroscopy, carbon 13 NMR, and heteronuclear multiple bond correlation data. This study showed that the bioactive compound isolated from C. microcarpa was 2-hydroxypropane-1,2,3-tricarboxylic acid monohydrate. The minimum inhibitory concentration （MIC） values of crude C. microcarpa extract and its bioactive component, 2-hydroxypropane-1,2,3-tricarboxylic acid as well as commercially available synthetic 2-hydroxypropane-1,2,3-tricarboxylic acid, were determined against 18 isolates of Edwardsiella tarda and 7 bacterial reference strains, namely, Escherichia coli （ATCC 25922）, Citrobacter freundii （ATCC 8090）, Aeromonas hydrophila （ATCC 49140）, Pseudomonas aeruginosa （ATCC 35032）, Streptococcus agalatiae （ATCC 13813）, E. tarda （ATCC 15947）, and Yersinia enterocolitica （ATCC 23715）, using two-fold microdilution method. The MIC values for both the natural 2-hydroxypropane-1,2,3-tricarboxylic acid and the synthetic one were ranging from 15.6 to 62.5 mg mL-1, whereas that of the crude extract was ranging from 7.8 to 31.3 mg mL-1. These findings showed that both the crude extract and its bioactive component might have potential as antimicrobial agent for aquaculture use.     

Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1 beta in solution

A method is presented that dramatically improves the resolution of protein nuclear magnetic resonance (NMR) spectra by increasing their dimensionality to four. The power of this technique is demonstrated by the application of four-dimensional carbon-13--nitrogen-15 (13C-15N)--edited nuclear Overhauser effect (NOE) spectroscopy to interleukin-1 beta, a protein of 153 residues. The NOEs between NH and aliphatic protons are first spread out into a third dimension by the 15N chemical shift of the amide 15N atom and subsequently into a fourth dimension by the 13C chemical shift of the directly bonded 13C atoms. By this means ambiguities in the assignment of NOEs between NH and aliphatic protons that are still present in the three-dimensional 15N-edited NOE spectrum due to extensive chemical shift overlap and degeneracy of aliphatic resonances are completely removed. Consequently, many more approximate interproton distance restraints can be obtained from the NOE data than was heretofore possible, thereby expanding the horizons of three-dimensional structure determination by NMR to larger proteins.     

Three-dimensional structure of the adenovirus major coat protein hexon

The three-dimensional crystal structure of the adenovirus major coat protein is presented. Adenovirus type 2 hexon, at 967 residues, is now the longest polypeptide whose structure has been determined crystallographically. Taken with our model for hexon packing, which positions the 240 trimeric hexons in the capsid, the structure defines 60% of the protein within the 150 X 10(6) dalton virion. The assembly provides the first details of a DNA-containing animal virus that is 20 times larger than the spherical RNA viruses previously described. Unexpectedly, the hexon subunit contains two similar beta-barrels whose topology is identical to those of the spherical RNA viruses, but whose architectural role in adenovirus is very different. The hexon structure reveals several distinctive features related to its function as a stable protective coat, and shows that the type-specific immunological determinants are restricted to the virion surface.     

塔拉多糖胶的研究

研究了塔拉豆的化学组成和塔拉多糖胶的化学结构及流变性质。塔拉豆中蛋白质含量为13.2%,胚乳占种子组成的30.5%。通过GC,PC,IR及NMR分析表明:胚乳中含有78.0%的半乳甘露聚糖,主链是以β-(1,4)-苷键连接的D-吡喃甘露糖,支链是以α-(1,6)-苷键连接的D-吡喃半乳糖,半乳糖与甘露糖配比为1:2.4。塔拉胶水溶液为假塑性流体,其1%水溶液表观粘度达313mPa·s(30℃,170S^-1),塔拉多糖胶与黄原胶或卡拉胶共混能形成凝胶。     

农产品安全检测仪器分析方法及其发展趋势

当前仪器分析已成为农产品安全检测的重要方法。常规分析仪器的检测方法主要包括色谱分析法、光谱分析法、高效毛细管电泳、生物传感技术等。农产品安全检测仪器分析方法的发展趋势主要表现为：（1）向高灵敏度、高选择性专用或广谱检测器发展;（2）向常压离子化质谱技术方向发展;（3）向仪器微型化、微分析和芯片技术方向发展。     

Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution

The 2.8 A resolution three-dimensional structure of a complex between an antigen (lysozyme) and the Fab fragment from a monoclonal antibody against lysozyme has been determined and refined by x-ray crystallographic techniques. No conformational changes can be observed in the tertiary structure of lysozyme compared with that determined in native crystalline forms. The quaternary structure of Fab is that of an extended conformation. The antibody combining site is a rather flat surface with protuberances and depressions formed by its amino acid side chains. The antigen-antibody interface is tightly packed, with 16 lysozyme and 17 antibody residues making close contacts. The antigen contacting residues belong to two stretches of the lysozyme polypeptide chain: residues 18 to 27 and 116 to 129. All the complementarity-determining regions and two residues outside hypervariable positions of the antibody make contact with the antigen. Most of these contacts (10 residues out of 17) are made by the heavy chain, and in particular by its third complementarity-determining region. Antigen variability and antibody specificity and affinity are discussed on the basis of the determined structure.     

Effects of pulse shaping in laser spectroscopy and nuclear magnetic resonance

Pulsed excitation fields are routinely used in most laser and nuclear magnetic resonance (NMR) experiments. In the NMR case, constant amplitude (rectangular) pulses have traditionally been used; in laser spectroscopy the exact pulse shape is often unknown or changes from shot to shot. This article is an overview of the effects of radio-frequency and laser pulse shapes and the instrumental requirements for pulse shaping. NMR applications to selective excitation, solvent suppression, elimination of phase roll, and reduced power dissipation are discussed, as are optical applications to soliton generation, velocity selective excitation, and quantitative population transfer.     

Tertiary structure is a principal determinant to protein deamidation

The protein deamidation process involves the conversion of the amide side-chain moieties of asparagine and glutamine residues to carboxyl groups. This conversion is an unusual form of protein modification in that it requires catalysis by an intramolecular reaction where both the substrate (asparagine and glutamine side chains) and "catalytic site" (the peptide nitrogen of the succeeding residue) are constituents of several consecutive residues along the polypeptide chain. The stereochemical factors governing this process were studied with a data base derived from the neutron crystallographic structure of trypsin from which amide groups and oxygen can be unambiguously differentiated because of their different neutron scattering properties. The neutron structure allowed for the direct determination of those residues that were deamidated; 3 of 13 asparagine residues were found to be modified. These modified residues were clearly distinguished by a distinct local conformation and hydrogen-bonding structure in contrast to those observed for the other asparagine residues. No correlation was found between preference to deamidate and the chemical character of residues flanking the site, as had been proposed from previous peptide studies.     

The nature of the metal-metal bond in bimetallic surfaces

The formation of a surface metal-metal bond can produce large perturbations in the electronic, chemical, and catalytic properties of a metal. Recent studies indicate that charge transfer is an important component in surface metal-metal bonds that involve dissimilar elements. The larger the charge transfer, the stronger the cohesive energy of the bimetallic bond. On a surface, the formation of a heteronuclear metal-metal bond induces a flow of electron density toward the element with the larger fraction of empty states in its valence band. This behavior is completely contrary to that observed in bulk alloys, indicating that the nature of a heteronuclear metal-metal bond depends strongly on the structural geometry of the bimetallic system.     

Noninvasive study of high-energy phosphate metabolism in human heart by depth-resolved 31P NMR spectroscopy

Phosphorus-31 nuclear magnetic resonance (NMR) spectra showing the relative concentrations of high-energy phosphate metabolites have been recorded noninvasively from the human heart in vivo. Spectral data were spatially localized by combining a pulsed magnetic field gradient with surface NMR excitation-detection coils. The location of the selected spectral region was determined by conventional proton NMR imaging immediately before examination by phosphorus-31 NMR spectroscopy.     

Evidence for a phytotoxic hydroxy-aluminum polymer in organic soil horizons

The toxicity of A1 that has been mobilized in soil, streams, and lakes through acid deposition primarily has been attributed to mononuclear A1 species. Polynuclear A1 species are more toxic than mononuclear species, but they have not been considered to be significant in the environment. Aluminum-27 nuclear magnetic resonance (NMR) spectra of forested spodosol soil horizon samples show the presence of polynuclear A1O(4)A1(12)(OH)(24)(H(2)O)(12)(7+). The AlO(4)A1(12)(OH)(24)(H(2)O)(12)(7+) species accounted for 30 percent of the aqueous A1 observable by NMR, and this could make a significant contribution to the toxicity of the A1 in these soils.     

己烯雌酚人工抗原的合成和酶免疫检测方法的建立

通过化学方法合成己烯雌酚单羧甲基醚,并通过核磁共振和质谱进行鉴定;分别用混合酸酐法和碳二亚胺法将其与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)偶联,合成己烯雌酚人工抗原,经紫外扫描、聚丙烯酰胺凝胶电泳和动物免疫试验证实人工抗原合成成功,并制备了抗己烯雌酚的特异性多克隆抗体,同时建立了己烯雌酚的酶免疫检测方法,这对动物性食品中己烯雌酚的残留检测有重要意义。     

二斑叶螨几丁质酶基因的克隆及特性分析

几丁质酶广泛存在于自然界的许多物种,并参与几丁质降解、蜕皮、免疫和致病性等诸多功能.采用同源克隆结合RACE-PCR的方法,首次从螨类中克隆到二斑叶螨几丁质酶基因(TuChi)的cDNA全长序列(GenBank登录号:JQ041366).TuChi全长1 822 bp,包含3'非翻译区(UTR)为150 bp和5'UTR为52 bp,开放阅读框(ORF)为1 620 bp,编码539个氨基酸.预测的相对分子量为60.7 ku,理论等电点为5.99.二斑叶螨几丁质酶具有几丁质酶典型的结构域,包括1个N-端信号肽序列、1个几丁质催化区域、1个几丁质结合域,以及连接催化区和结合域之间的连接区域.与其他物种几丁质酶进化树比较分析表明,TuChi与昆虫几丁质酶家族Group 1具有较高的同源性,推测其与二斑叶螨的蜕皮密切相关.     

Mg/Al ordering in layered double hydroxides revealed by multinuclear NMR spectroscopy

The anion-exchange ability of layered double hydroxides (LDHs) has been exploited to create materials for use in catalysis, drug delivery, and environmental remediation. The specific cation arrangements in the hydroxide layers of hydrotalcite-like LDHs, of general formula Mg2+(1-x)Al3+(x)OH2(Anion(n-)(x/n)).yH2O, have, however, remained elusive, and their elucidation could enhance the functional optimization of these materials. We applied rapid (60 kilohertz) magic angle spinning (MAS) to obtain high-resolution hydrogen-1 nuclear magnetic resonance (1H NMR) spectra and characterize the magnesium and aluminum distribution. These data, in combination with 1H-27Al double-resonance and 25Mg triple-quantum MAS NMR data, show that the cations are fully ordered for magnesium:aluminum ratios of 2:1 and that at lower aluminum content, a nonrandom distribution of cations persists, with no Al3+-Al3+ close contacts. The application of rapid MAS NMR methods to investigate proton distributions in a wide range of materials is readily envisaged.     

The impacts of oxytetracycline on humification during manure composting can be alleviated by adjusting initial moisture contents as illustrated by NMR

Oxytetracycline(OTC) residues have been found in soil and water, and they may pose potential risks to agricultural ecological environments. One of the most impactful ways for OTC to enter the soil and water environments is through excrement used as organic fertilizer. Therefore, it is important to remove OTC during manure composting and to understand the transformation of the organic materials during composting in the presence of OTC. In the present paper, chicken manure and wheat sawdust spiked with OTC were composted under different initial moisture contents(MC) to evaluate the degradation of OTC and changes of organic matter during the composting process. The MC has a significant effect on OTC degradation during composting. A higher MC of 65% was more conducive to OTC degradation(77.4%) and compost maturity compared to the lower MC. However, the higher MC of 65% could increase the ammonia volatilization by promoting nitrification compared to the lower MC. An increase in the initial MC could improve the composting temperature. NMR results illustrated that the presence of OTC could affect the internal transformation of aliphatics, and OTC inhibited compost humification. Thus, an initial MC of 55–65% can alleviate the impacts of OTC on humification during manure composting.