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1.
The vacuum-ultraviolet circular dichroism (VUVCD) spectra of various amino acids, saccharides, and proteins were measured using a synchrotron-radiation CD spectrophotometer at HiSOR/HSRC that is capable of measuring the CD spectra down to 140 nm in aqueous solution. L-Isomers of amino acids show two successive positive peaks at around 200 and 180 nm depending on the side chain. The ab initio assignment by time-dependent density functional theory predicts that these peaks are attributed to n-pi* and pi-pi* transitions of the carboxyl group, respectively. Most mono- and disaccharides exhibit characteristic peaks at around 170 nm, sensitively depending on the anomeric and axial/equatorial configurations of hydroxyl groups, trans-gauche conformations of the hydroxymethyl group, and the type of glycosidic linkage. The VUVCD spectra of 31 globular proteins allow us to estimate more accurately the content and number of alpha-helix and beta-strand segments by extending the short-wavelength limit of the analytical program SELCON3 down to 160 nm. These results demonstrate that synchrotron-radiation VUVCD spectroscopy is a useful tool for structure analyses of biomolecules in solution based on the higher energy transitions of chromophores. 相似文献
2.
The vacuum ultraviolet circular dichroism (VUVCD) spectra of 15 globular proteins (myoglobin, hemoglobin, human serum albumin, cytochrome c, peroxidase, alpha-lactalbumin, lysozyme, ovalbumin, ribonuclease A, beta-lactoglobulin, pepsin, trypsinogen, alpha-chymotrypsinogen, soybean trypsin inhibitor, and concanavalin A) were measured in aqueous solutions at 25 degrees C in the wavelength region from 260 to 160 nm under a high vacuum, using a synchrotron-radiation VUVCD spectrophotometer. The VUVCD spectra below 190 nm revealed some characteristic bands corresponding to different secondary structures. The contents of alpha-helices, beta-strands, turns, and unordered structures were estimated using the SELCON3 program with VUVCD spectra data on the 15 proteins. Prediction of the secondary-structure contents was greatly improved by extending the circular dichroism spectra to 165 nm. The numbers of alpha-helix and beta-strand segments calculated from the distorted alpha-helix and beta-strand contents did not differ greatly from those obtained from X-ray crystal structures. These results demonstrate that synchrotron-radiation VUVCD spectroscopy is a powerful tool for analyzing the secondary structures of proteins. 相似文献
3.
The vacuum-ultraviolet circular dichroism (VUVCD) spectra of 16 globular proteins (insulin, lactate dehydrogenase, glucose isomerase, lipase, conalbumin, transferrin, catalase, subtilisin A, alpha-amylase, staphylococcal nuclease, papain, thioredoxin, carbonic anhydrase, elastase, avidin, and xylanase) were successfully measured in aqueous solutions at 25 degrees C from 260 to 160 nm under a high vacuum using a synchrotron-radiation VUVCD spectrophotometer. These proteins exhibited characteristic CD spectra below 190 nm that were related to their different secondary structures, which could not be detected with a conventional CD spectrophotometer. The component spectra of alpha-helices, beta-strands, turns, and unordered structures were obtained by deconvolution analysis of the VUVCD spectra of 31 reference proteins including the 15 proteins reported in our previous paper [Matsuo, K. et al. (2004) J. Biochem. 135, 405-411]. Prediction of the secondary-structure contents using the SELCON3 program was greatly improved, especially for alpha-helices, by extending the short-wavelength limit of CD spectra to 160 nm and by increasing the number of reference proteins. The numbers of alpha-helix and beta-strand segments, which were calculated from the distorted alpha-helix and beta-strand contents, were close to those obtained on X-ray crystallography. These results demonstrate the usefulness of synchrotron-radiation VUVCD spectroscopy for the secondary structure analysis of proteins. 相似文献
4.
Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy
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To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures. 相似文献
5.
Falconi M Bozzi M Paci M Raudino A Purrello R Cambria A Sette M Cambria MT 《International journal of biological macromolecules》2001,29(3):161-168
The interaction between monomeric insulin and monosaccharides has been investigated through circular dichroism, fluorescence spectroscopy and two dimensional nuclear magnetic resonance. CD spectra indicate that D-glucose interacts with monomeric insulin whereas D-galactose, D-mannose and 2-deoxy-D-glucose have a lower effect. Fluorescence emission was quenched at sugar concentrations of 5-10 mM. Titration with the different sugars produces a quenching of the tyrosine spectrum from which a binding free energy value for the insulin-sugar complexes has been evaluated. Transfer nuclear Overhauser enhancement NMR experiments indicate the existence of dipolar interactions at short interatomic distances between C-1 proton of D-glucose in the beta form and the monomeric insulin. Further, NMR total correlation spectra experiments revealed that the hormone is in the monomeric form and that upon addition of glucose no aggregation occurs. The interaction does not involve relevant changes in the secondary structure of insulin suggesting that the interaction occur at the side chain level. Molecular dynamics simulations and modeling studies, based on the dynamic fluctuations of potential binding moiety sidechains, argued from results of NMR spectroscopy, provide additional informations to locate the putative binding sites of D-glucose to insulin. 相似文献
6.
A number of complexes are synthesised with Pr(III) and Nd(III) employing the mono- and disaccharides, D-glucose, D-galactose, D-mannose, D-arabinose, D-ribose, D-xylose, maltose and lactose. The complexes are characterised by various spectral and analytical techniques, and based on these data, appropriate structures were assigned for all complexes. 相似文献
7.
A series of nine Ce(III) complexes has been synthesised with seven different monosaccharides (D-glucose, D-fructose, D-galactose, D-mannose, L-sorbose, D-ribose and D-xylose) and two different disaccharides (D-maltose and L-lactose), and these have been characterised with various analytical, spectral, magnetic and electrochemical techniques. The NMR studies have highlighted some interesting features about the metal-ion-binding pattern of the saccharides. Some additional coordination has been proposed along with the chelating groups in the saccharide molecules, based on the shifts in 13C NMR spectra. On the other hand, solution absorption studies and solid-state magnetic susceptibilities have indicated the contribution from the d-character to the spectral features to some extent. 相似文献
8.
An X-ray crystallographic study has confirmed that the potassium bisulfite adducts of D-glucose and D-mannose have open-chain structures with R and S configurations respectively at C-1. NMR studies have shown that each sugar gives rise to two bisulfite compounds, and solution-state structures and conformations of these isomers have been deduced from analysis of (1)H NMR spectra. (13)C NMR data for the four adducts are given. Furanose forms of the D-glucose and D-mannose have been detected in the equilibrium solutions. 相似文献
9.
Spruce and ash wood were subjected to acid hydrolysis. The hydrolysates were paper chromatographed for the presence of saccharides. The following monosaccharides were detected: D-xylose, D-mannose, D-glucose, D-galactose and L-arabinose. The monosaccharides and L-rhamnose in addition in the form of concentrated solutions were used as part of lactose in production of penicillin V. Similarly, the whole amounts of D-glucose and saccharose were substituted in biosynthesis of cephalosporin C. With regard to the results it follows that hydrolysates of the conifers and especially of the hardwood can substitute part of the fermentation medium components in production of penicillin V and cephalosporin C. 相似文献
10.
Circular dichroism studies of an oligo-alpha-thymidylate and of its interactions with complementary sequences. 总被引:3,自引:3,他引:0
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An octathymidylate synthesized with the alpha anomer of thymidine has been studied using circular dichroism. Its conformation has been compared to that of its analogue containing the naturally occurring beta anomer. In both compounds some degree of intramolecular stacking is present as indicated by the shapes of the circular dichroism spectra and their variations with temperature. As its beta-analogue the alpha-octathymidylate binds to its complementary sequences containing beta-nucleosides. Only complexes with 1A:1T stoechiometry were observed. Binding to ribose- containing oligomers and polymers is much stronger than binding to deoxyribose-containing analogues. Circular dichroism spectra provided evidence for a difference between the geometry of the various complexes formed with an alpha-oligothymidylate and those formed with its beta-anomer-containing analogue. 相似文献
11.
Partial characterization of Pseudomonas phage 2 receptor 总被引:1,自引:0,他引:1
F J Castillo 《Canadian journal of microbiology》1980,26(8):1015-1017
The lipopolysacharide from Pseudomonas aeruginosa strain BI contains the receptors for phage 2 and strongly inactivates this phage in vitro (95-98% within 15 min). Several mono- and di-saccharides tested reduced phage 2 inactivation to 50% when present at the following concentrations: D-glucosamine, 0.25 M; maltose, 0.3M; lactose and cellobiose, 0.5 M; D-glucose, L-rhamnose, D-mannose, 2-deoxy-D-glucose, and sucrose, 1.0 M; D-galactose, D-xylose, and N-acetyl-D-glucosamine, 1.4 M; and melibiose. greater than 1.6 M. These results suggest the possibility that phage 2 receptors in lipopolysaccharide contain L-rhamnose, D-glucosamine, and (or) D-glucose, or a structurally related molecule. Either one of the latter two could be located at a terminal position alpha-linked to the adjacent residue, or located internally in the polysaccharide chain linked through its C-4 position. 相似文献
12.
The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the alpha and beta anomers of these disaccharides, except those containing N-sulfated GlcN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the alpha anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3 ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15 ppm, respectively, but that of C-1 was shifted downfield by 0.25 ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of delta GlcA (D-gluco-4-ene-pyranosyluronic acid), the signal of the H-3 proton of delta GlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15 ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive. 相似文献
13.
T N Druzhinina N A Kalinchuk V N Shibaev N K Kochetkov 《Bioorganicheskaia khimiia》1988,14(11):1586-1588
A partial purified polymerase from S. anatum was used for the synthesis of polysaccharide [-6) [14C]Man(beta 1-4)Rha(alpha 1-3)Gal(alpha 1-]n and its analogues containing D-glucose residue instead of D-galactose or D-mannose. Structures of these polymers were confirmed by methylation analysis and radioimmunochemical tests. 相似文献
14.
Sensitivity of a Bacteroides melaninogenicus strain to monosaccharides: effect on enzyme induction. 总被引:3,自引:1,他引:2
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The inhibition of growth in Bacteroides melaninogenicus by sugars in described. Monosaccharides such as D-glucose, D-galactose, D-mannose, and D-fructose are inhibitory at low concentrations, whereas the disaccharides sucrose and lactose are not inhibitory even at high concentrations. The major inhibitory effect of the sugar is found during the transition of lag to logarithmic growth phases. There was no primary effect of D-glucose on protein, ribonucleic acid, or deoxyribonucleic acid synthesis on cells in transition from lag to logarithmic growth. However, the addition of glucose or galactose completely abolished the induction of 3-ketodihydrosphingosine synthetase by vitamin K in vitamin K-depleted cells. Futhermore, in cells which were not vitamin K depleted, the level of this enzyme was drastically reduced by the addition of the sugar. Cyclic adenosine 5-monophosphate was unable to reverse the growth inhibition produced by glucose. In actively growing cultures, addition of sugar slows the growth rate. In these experiments the level of 3-ketodihydrosphingosine synthetase fell only after the cells had assumed the slower rate of growth. There were two indications that D-galactose was more inhibitory than D-glucose; in the presence of 0.1% D-galactose cells in lag phase did not show the increase in turbidity found in similar cells placed in medium with 0.1% D-glucose, and also D-galactose caused a greater decrease in the growth rate of actively growing cultures than was found with D-glucose. These studies suggest that the inhibitory effect of monosaccharides in lag leads to logarithmic growth transition can be ascribed to an effect on enzyme induction. On the other hand, the ability of many monosaccharides to inhibit growth, and the greater inhibitory property of D-galactose compared with D-glucose, suggests that other mechanisms may be operative as well. 相似文献
15.
Synchrotron-radiation vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy can significantly improve the predictive accuracy of the contents and segment numbers of protein secondary structures by extending the short-wavelength limit of the spectra. In the present study, we combined VUVCD spectra down to 160 nm with neural-network (NN) method to improve the sequence-based prediction of protein secondary structures. The secondary structures of 30 target proteins (test set) were assigned into alpha-helices, beta-strands, and others by the DSSP program based on their X-ray crystal structures. Combining the alpha-helix and beta-strand contents estimated from the VUVCD spectra of the target proteins improved the overall sequence-based predictive accuracy Q(3) for three secondary-structure components from 59.5 to 60.7%. Incorporating the position-specific scoring matrix in the NN method improved the predictive accuracy from 70.9 to 72.1% when combining the secondary-structure contents, to 72.5% when combining the numbers of segments, and finally to 74.9% when filtering the VUVCD data. Improvement in the sequence-based prediction of secondary structures was also apparent in two other indices of the overall performance: the correlation coefficient (C) and the segment overlap value (SOV). These results suggest that VUVCD data could enhance the predictive accuracy to over 80% when combined with the currently best sequence-prediction algorithms, greatly expanding the applicability of VUVCD spectroscopy to protein structural biology. 相似文献
16.
To elucidate the structural characteristics of alcohol-denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra of six proteins-myoglobin, human serum albumin, α-lactalbumin, thioredoxin, β-lactoglobulin, and α-chymotrypsinogen A-down to 170 nm in trifluoroethanol solutions (TFE: 0-50%) and down to 175 nm in methanol solutions (MeOH: 0-70%) at pH 2.0 and 25°C, using a synchrotron-radiation VUVCD spectrophotometer. The contents of α-helices, β-strands, turns, poly-L-proline type II helices (PPIIs), and unordered structures of these proteins were estimated using the SELCON3 program, including the numbers of α-helix and β-strand segments. Furthermore, the positions of α-helices and β-strands on amino acid sequences were predicted by combining these secondary-structure data with a neural-network method. All alcohol-denatured proteins showed higher α-helix contents (up to ~ 90%) compared with the native states, and they consisted of several long helical segments. The helix-forming ability was higher in TFE than in MeOH, whereas small amounts of β-strands without sheets were formed in the MeOH solution. The produced α-helices were transformed dominantly from the β-strands and unordered structures, and slightly from the turns. The content and mean length of α-helix segments decreased as the number of disulfide bonds in the proteins increased, suggesting that disulfide bonds suppress helix formation by alcohols. These results demonstrate that alcohol-denatured proteins constitute an ensemble of many long α-helices, a few β-strands and PPIIs, turns, and unordered structures, depending on the types of proteins and alcohols involved. 相似文献
17.
The relative contribution of each anomer of D-glucose to the overall phosphorylation rate of the hexose tested at anomeric equilibrium was examined in rat liver postmicrosomal supernatants under conditions aimed at characterizing the activity of glucokinase, with negligible interference of either hexokinase, N-acetyl-D-glucosamine kinase or glucose-6-phosphatase (acting as a phosphotransferase). Both at 10 degrees and 30 degrees C, the relative contribution of each anomer was unaffected by the concentration of D-glucose. At both temperatures, the alpha/beta ratio for the contribution of each anomer was slightly, but significantly, lower than the alpha/beta ratio of anomer concentrations. These findings, which are consistent with the anomeric specificity of glucokinase in terms of affinity, cooperativity and maximal velocity, reveal that the preferred alpha-anomeric substrate for both glycogen synthesis and glycolysis is generated by glucokinase at a lower rate than is beta-D-glucose-6-phosphate. 相似文献
18.
C D Warren I Y Liu A Herscovics R W Jeanloz 《The Journal of biological chemistry》1975,250(20):8069-8078
2,3,4,6-Tetra-O-acetyl-beta-D-mannopyranosyl phosphate, free of the alpha anomer, was coupled with citronellol and dolichol in the presence of triisopropylbenzenesulfonyl chloride to give, after chromatographic purification and deacetylation, the respective polyisoprenyl beta-D-mannopyranosyl phosphates. These compounds were compared with the previously synthesized alpha anomers by means of their chromatographic properties, spectra, optical rotations, and hydrolysis reactions when treated with acid and alkali. To characterize the compounds resulting from these treatments, and to determine the mechanism of the alkaline hydrolysis, beta-D-mannopyranosyl phosphate was converted into beta-D-mannonpyranose 1,2-phosphate, and hence into D-mannose 2-phosphate, obtained as a mixture of alpha and beta anomers, characterized by infrared and nuclear magnetic resonance spectra and elemental analysis. Beta-D-Mannopyranosyl phosphate was readily separated by thin layer chromatography from the corresponding alpha anomer. 相似文献
19.
The specificity of Saccharomyces cerevisiae yeast on the removal of carbohydrates by fermentation was studied. The common monosaccharides, D-glucose, D-fructose, D-mannose, and D-galactose were completely removed; D-glucuronic acid and D-ribose were partially removed; but D-xylose, D-rhamnose, and L-sorbose were not removed and were completely resistant. Of four glycosides, methyl and phenyl alpha- and beta-D-glucopyranosides, three of the four were partially removed and methyl beta-D-glucopyranoside was not removed. The disaccharides, maltose, sucrose, and turanose were completely removed, while cellobiose, lactose, and melibiose were completely resistant. Isomaltose and alpha,alpha-trehalose were partially removed. Maltotriose and raffinose were partially removed, but isomaltotriose and melezitose were completely resistant. The tetrasaccharides, maltotetraose, isomaltotetraose, and acarbose, were completely resistant. Further, the yeast enzymes did not alter any of the resistant carbohydrates by transglycosylation or condensation reactions or by any other types of reactions. 相似文献
20.
The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of valency hybrid hemoglobins and their constituents (alpha + and beta chains for alpha 2+beta 2, alpha and beta + chains for alpha 2 beta 2+: + denotes ferric heme) were measured in the Soret region for F-, H2O, N3- and CN- derivatives. Absorption and MCD spectra of valency hybrid hemoglobins were very similar to the arithmetic mean of respective spectra of their corresponding component chains in all derivatives. The Soret MCD intensity around 408 nm for various complexes of valency hybrid hemoglobins seems to reflect the spin state of ferric chains. Upon ferric and deoxy ferrous subunit association to make the deoxy valency hybrid hemoglobins, only the high-spin forms bound with F- and H2O of alpha 2+beta 2 displayed a blue shift in the peak position around 430 nm and those of alpha 2 beta 2+ an increase in intensity around 430 nm. The blue shift and the increase in intensity were considered to be caused by the structural changes in deoxy beta chains of alpha 2+beta 2 and deoxy alpha chains of alpha beta 2+, respectively. These spectral changes were interpreted on the basis of their oxygen-equilibrium properties. In contrast to absorption and MCD spectra, the CD spectra of valency hybrid hemoglobins were markedly different from the simple addition of those of their component chains in all derivatives examined. The large part of CD spectral changes upon subunit association were interpreted as changes in the heme vicinity accompanied by formation of the alpha 1 beta 1 subunit contact. 相似文献