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1.
Cobrotoxin (Mr 6949), which binds tightly to the acetylcholine receptors, contains no phenylalanines and only two histidines, two tyrosines, and one tryptophan that result in well-resolved aromatic proton resonances in D2O at 360 MHz. His-32, Tyr-25, and the Trp are essential for toxicity and may interact with the acetylcholine receptor. We assign two titratable resonances (pKa = 5.1) at delta = 9.0 and 7.5 ppm at pH 2.5 and at 7.7 and 7.1 ppm at pH 9.5 to the C-2 and C-4 ring protons, respectively, of His-4. Two other titratable resonances (pKa = 5.7) at delta = 8.8 and 6.9 ppm at pH 2.5 and at 7.8 and 6.7 ppm at pH 9.5 are assigned to the C-2 and C-4 ring protons of His-32, respectively. The differences in delta values of the two histidines reflect chemically different microenvironments while their low pKa values could arise from nearby positive charges. A methyl resonance gradually shifts upfield to delta approximately 0.4 ppm as His-4 is deprotonated and is tentatively assigned to the methyl group of Thr-14 or Thr-15 which, from published X-ray studies of neurotoxins, are located in the vicinity of His-4. Further, we have identified the aromatic resonances of the invariant tryptophan and individual tyrosines and the methyl resonance of one of the two isoleucines in the molecule. Several broad nontitrating resonances of labile protons which disappear at pH greater than 9 may arise from amide groups of the beta sheet in cobrotoxin. 相似文献
2.
We have studied the nmr spectra of the series of alanine oligopeptides containing a methoxyethoxyethoxyacetyl blocking group on the N-terminal residue and a morpholino blocking group on the C-terminal residue. Spectra were measured in chloroform–trifluoroacetic acid solvent systems. For oligomers with chain lengths of five or more, “double peaks” are observed for the α-CH protons. Addition of trifluoroacetic acid causes the peaks to coalesce. The amount of trifluoroacetic acid necessary for coalescence increases from the pentamer to the nonamer. These findings are general since alanine oligomers with different blocking groups exhibit similar “double peak” phenomena. We explain the “double peak” phenomenon in terms of specific folded forms of the oligopeptides which arise from intramolecular hydrogen bonding. Additional evidence for such hydrogen bonding is presented based upon infrared studies. Slight aggregation probably occurs for the pentamer and hexamer which may stabilize the folded forms. 相似文献
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High-resolution 270-MHz proton nuclear magnetic resonance (NMR) spectra of the native two-zinc insulin hexamer at pH 9 have been obtained, and assignments of key resonances have been made. Spectra of zinc-free insulin titrated with Zn2+ are unchanged after the addition of 1 equiv of zinc per insulin hexamer, indicating that the conformation of the hexamer is fixed at this point and that the second zinc ion does not significantly change the conformation. Titration of the two-zinc insulin hexamer with anions high on the Hofmeister series such as SCN- causes marked changes in the NMR spectra which are interpreted as the result of major conformational changes to a new hexameric form of insulin having a twofold axis perpendicular to the threefold axis. Analysis of difference spectra indicates that this new hexamer (which should be capable of binding six zinc ions) binds 2 equiv of SCN- at two sites which are assumed to be identical and independent (K1 = 10(3), K2 = 2.5 X 10(2) M-1). 相似文献
5.
Conformational characterization of ceramides by nuclear magnetic resonance spectroscopy 总被引:1,自引:0,他引:1 下载免费PDF全文
Ceramide (Cer) has been identified as an active lipid second messenger in the regulation of cell growth, differentiation, and apoptosis. Its analog, dihydroceramide, without the 4 to 5 trans double bond in the sphingoid backbone lacks these biological effects. To establish the conformational features that distinguish ceramide from its analogs, nuclear magnetic resonance spectral data were acquired for diluted samples of ceramides (C2- and C18-Cer), dihydroceramide (C16-DHCer), and deoxydihydroceramide (C18-DODHCer). Our results suggest that in both C2- and C18-Cer, an H-bond network is formed in which the amide proton NH is donated to the OH groups on carbons C1 and C3 of the sphingosine backbone. Two tightly bound water molecules appear to stabilize this network by participating in flip-flop interactions with the hydroxyl groups. In DHCer, the lack of the trans double bond leads to a conformational distortion of this H-bonding motif. Without the critical double bond, the degree with which water molecules stabilize the H bonds between the two OH groups of the sphingolipid is reduced. This structural alteration might preclude the participation of DHCer in signaling-related interactions with cellular targets. 相似文献
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This paper presents the first 1H-NMR spectra of the aromatic region of adrenodoxin, a mammalian mitochondrial 2Fe-2S non-heme iron ferredoxin. One-dimensional proton NMR spectra of both reduced and oxidized adrenodoxin were recorded as a function of pH. Resonances due to two of the three histidines of adrenodoxin gave sharp signals in the one-dimensional proton NMR spectra. The pKa values of the resolved histidine resonances in the oxidized protein were 6.64 +/- 0.03 and 6.12 +/- 0.06. These values were unchanged when adrenodoxin was reduced by the addition of sodium dithionite. In addition, the oxidized protein showed a broadened histidine C-2H resonance with a pKa value of approx. 7. This resonance was not apparent in the spectra of the reduced protein. The resonances due to the single tyrosine in adrenodoxin were identified using convolution difference spectroscopy. In addition, a two-dimensional Fourier-transform double quantum filtered (proton, proton) chemical shift correlated (DQF-COSY) spectrum of oxidized adrenodoxin was obtained. The cross peaks of the resonances due to the tyrosine, the four phenylalanines, and two of the three histidines of adrenodoxin were resolved in the DQF-COSY spectrum. Reduction of the protein caused several changes in the aromatic region of the NMR spectra. The resonances assigned to the C2 proton of the histidine with a pKa of 6.6 shifted upfield approx. 0.15 ppm. In addition, when the protein was reduced one of the resonances assigned to a phenylalanine residue with a chemical shift of 7.50 ppm appeared to move downfield to 7.82 ppm.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Proton correlation nuclear magnetic resonance has been used to investigate anaerobic metabolism of glucose in Escherichia coli cells. The time course of the concentrations of six metabolites (ethanol, lactate, acetate, pyruvate, succinate, and formate) has been followed at the very early state of fermentation, and used to discuss dynamical aspects of the mixed-acid fermentation of glucose by E. coli. 相似文献
11.
A proton nuclear magnetic resonance (NMR) study is reported of human alpha-2-macroglobulin (alpha-2-M). It was observed that alpha-2-M, which consists of four identical subunits and has a molecular weight of 720,000, gives several sharp resonances. After cleavage of the "bait" region peptide with trypsin and subsequent removal of the peptide under a high salt condition, most of the sharp resonances disappeared, indicating that the sharp resonances observed in the native alpha-2-M originate from the amino acid residues in the bait region. Resonances due to the aromatic protons of the Tyr residue, which exists in the bait region, have been assigned on the basis of chemical shift. It was observed that the C3- and C5-H proton resonances for the Tyr residue are especially narrow, indicating that the side chain of the Tyr residue in the bait region is in a highly mobile state. Photochemically induced dynamic nuclear polarization experiments clearly show that the Tyr residue is actually exposed to the solvent. It was possible to identify resonances due to several His residues that are exposed to solvent. Other resonances, which probably originate from Arg residues in the bait region, were also observable in the conventional NMR spectra. On the basis of the present NMR data, we conclude that the bait region of the native alpha-2-M is highly flexible and exposed to solvent. On treatment of alpha-2-M with methylamine, no significant change has been detected in the NMR spectra observed in both the conventional and CIDNP mode.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
G M Smith 《Biochemistry》1979,18(8):1628-1634
Rhodospirillum rubrum cytochrome c2 was studied by proton nuclear magnetic resonance at 220 MHz. Assignments were made to the resonances of heme c by double-resonance techniques and by temperature-dependence studies. The aromatic resonances of Trp-62 and Tyr-70 of ferrocytochrome c2 were identified by spin-decoupling experiments. The resonances of the Met-91 methyl group of the ferri- and ferrocytochromes were assigned by saturation-transfer experiments. The assignments are compared to those made for cytochromes c. A pH titration showed that the methionine methyl resonance of ferricytochrome c2 shifted with a pK of 6.25 and disappeared above pH 9. No histidine CH resonances that titrated normally over the neutral pH range were observed in the spectrum of either oxidation state of the protein. The possible origins of the ionizations at pH 6.25 and 9 are discussed. 相似文献
13.
The low-field 220-MHz proton nuclear magnetic resonance (NMR) spectra of four tRNA molecules, Escherichia coli tRNAPhe, tRNA1Val, and tRNAfMet1, and yeast tRNAPhe, at neutral and mildly acidic pH are compared. We find a net increase in the number of resonances contributing to the -9.9-ppm peak (downfield from sodium 4,4-dimethyl-4-silapentanesulfonate) in three of these tRNAs at pH 6, while tRNAfMet1 does not clearly exhibit this behavior. The increase in intensity at this resonance position is half-completed at pH 6.2 in the case of yeast tRNAPhe. An alteration at the 5'-phosphate terminus is not involved, since removal of the terminal phosphate does not affect the gain in intensity at -9.9 ppm. Based on a survey of the tertiary interactions in the four molecules, assuming that they possess tertiary structures like that of yeast tRNAPhe at neutral pH, we tentatively attribute this altered resonance in E. coli and yeast tRNAPhe to the protonation of the N3 of the adenine residue at position 9 which results in the stabilization of the tertiary triple A23-U12-A9. This intepretation is supported by model studies on the lowfield proton NMR spectrum of AN oligomers at acid pH, which reveal an exchanging proton resonance at -9.4 ppm if the chain length N greater than or equal to 6. 相似文献
14.
Proton decoupled fluorine nuclear magnetic resonance spectroscopy in situ. 总被引:1,自引:0,他引:1 下载免费PDF全文
The efficacy of proton decoupling for enhancing the 19F nuclear magnetic resonance (NMR) signal-to-noise ratio and spectral resolution in the intact subject is demonstrated. A geometrically orthogonal cross-coil antenna configuration (Helmholtz pair, surface coil) is employed to provide 40 dB of isolation between the 19F observe and 1H decouple frequencies of 188 and 200 MHz, respectively. Further isolation is achieved through the use of high-quality notch filters on both observation and decoupling channels. Application of 19F-(1H) NMR spectroscopy to the study of 2-fluoro-2-deoxy-D-glucose metabolism in cerebral tissue in situ is presented. Significant improvements in sensitivity and resolution are obtained and result from both a collapse of the JFH multiple structure and a substantial positive nuclear Overhauser effect (NOE). To our knowledge, this is the first such demonstration of 1H decoupling in conjunction with 19F observation for study of the metabolism of a fluorinated compound in the living subject. 相似文献
15.
High resolution proton nuclear magnetic resonance (NMR) spectra of normal and diseased human muscle extracts were recorded at 470 MHz. Resonances from lactic acid, creatine, glucose, ribose, purine and pyrimidine bases were identified. The longitudinal relaxation times of these resonances were determined to allow quantitation of muscle metabolites. With aid of a standardized reference capillary, inserted into the NMR tube containing the muscle extracts, the lactic acid and total creatine content of the extracts was determined. After 5 h of incubation at 37 degrees C, normal muscles contained on average 103 mumol lactic acid and 36 mumol creatine/173 mg of noncollagenous protein, equivalent to 1.0 g of fresh muscle. The lactic acid and creatine contents decreased slightly in scoliosis and idiopathic scoliosis and they decreased significantly in cerebral palsy. In an extract of a patient whose illness was diagnosed as 'scoliosis' no creatine was present, and in an extract of a patient with unknown diagnosis the creatine content was reduced to 2 mumol/173 mg of noncollagenous protein. The short time (1.7 sec to 6.5 min) and the small amount of tissue (300 mg) needed for an analysis add to the potential of proton NMR as a new technique for the characterization of muscular diseases. 相似文献
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The aqueous solution conformation of Tyr-Asn-Ile-Gln-Lys (UB5) corresponding to positions 59-63 of the polypeptide, ubiquitin, has been investigated by proton NMR. Like the parent protein, UB5 induces nonspecifically both T and B lymphocyte differentiation. The various NH and CH resonances of this pentapeptide have been assigned, and its solution conformation has been probed through a study of chemical shift variations with pH, temperature dependence of amide hydrogen chemical shifts, vicinal NH--C alpha H and C alpha H--C beta H2 coupling constant data, and amide hydrogen-exchange rates. The latter were measured in H2O by using a combination of transfer of solvent saturation and saturation recovery NMR experiments. The data are compatible with the assumption of a highly motile dynamic equilibrium among different conformations for this peptide. The various secondary amide hydrogens remain essentially exposed to the solvent. The temperature-dependence study of the amide hydrogen chemical shifts also did not reveal any strong internal hydrogen bonds. A rotamer population analysis of tyrosine and asparagine side chains suggests that two of the rotomers are predominantly populated for each of these residues. From these results, a picture emerges of the dynamic conformation of UB5 in aqueous solution. 相似文献
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A 1H nuclear magnetic resonance (NMR) study was carried out on various ferredoxins which possess one of three types of iron-sulfur clusters, (2Fe-2S), (3Fe-3S), or (4Fe-4S). In the isolated form, (2Fe-2S) ferredoxins from spinach (Spinacea oleracia), pokeweed (Phytolacca americana), a blue-green alga (Spirulina platensis), and a halobacterium (Halobacterium halobium) exhibited two broad resonances common in chemical shift at the region downfield of 10 ppm. In their reduced forms, seven contact-shifted resonances appeared spread over 30 ppm. Although the positions of the contact-shifted resonances in the reduced state differed among the four, a common trend in the temperature dependence of their resonance positions was recognized. Two (4Fe-4S) ferredoxins from Bacillus stearothermophilus and Bacillus thermoproteolyticus exhibited almost indistinguishable spectral patterns in both the oxidized and reduced forms. The ferricyanide-treated ferredoxins of B. stearothermophilus and B. thermoproteolyticus showed characteristic contact-shifted resonances distinct from the spectra of the original (4Fe-4S) ferredoxins. This corresponds to the recent finding of the interconversion of (4Fe-4S) and (3Fe-3S) clusters with ferricyanide in the ferredoxin. Based on our data together with reported NMR data on other ferredoxins, contact-shift resonances of three types of clusters were tabulated. The reliability of NMR classification increases when we compare the NMR spectra of a ferredoxin with the classification standards at the two redox states. Moreover, not only the absolute values of the chemical shifts of contact-shifted resonances but also their temperature dependence give distinctive information applicable to iron core identification. 相似文献
19.
Bovine, porcine and sheep adrenodoxin, and the trypsin-resistant form of bovine adrenodoxin have been studied by one- and two-dimensional 1H-NMR spectroscopy. Assignment of the resonances for all the aromatic amino acids with resolved aromatic resonances have been made by correlating NMR spectra with the amino acid sequences from various species. Slowly exchanging amide protons and downfield shifted alpha-protons of His10 and Phe11 suggest possible involvement in beta-sheet structure. The effects on the assigned resonances due to the specific spin-label with a nitroxide radical at Cys95 have been analyzed on a two-dimensional 1H-NMR spectrum. The present results provide evidence for a structural similarity with a model for the structure of adrenodoxin based on a sequence alignment with that of Spirulina platensis ferredoxin, for which X-ray crystallographic data is available. epsilon-Methyl groups of Met120 and Met122 have been assigned by comparing 1H-NMR spectra of adrenodoxin with those of the trypsin-resistant form of adrenodoxin which is specifically cleaved at Arg115. epsilon-Methyl groups of Met120 and Met122 have an exceptionally long longitudinal relaxation time compared with those of valyl and leucyl methyl groups, suggesting that the COOH-terminal peptide spanning over 13 amino acids rotates rather freely in the solvent. 相似文献