Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:Q9UMR3 (NMR)
 150,598 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of "solvent-exposed" tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cgamma of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 +/- 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. (i) Peak chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 coordinated to the metal. (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either. (iv) The gamma carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.
 ...
PMID:Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy. 1 66

Using a combination of ultraviolet-visible absorption, 1H NMR and ESR techniques we have established that N(1) of the imidazole and N(1) of the pyrimidine residues of bleomycin A2 bind to Cu(II) and Zn(II). The observations coupled with the earlier results that the alpha-amino group of the alpha-amino carboxamide function and the carbamoyl moiety are also Cu(II)-ligating groups makes it possible to reconstruct the detailed geometry and stereochemistry of the metal binding site of bleomycin A2.
 ...
PMID:A spectroscopic investigation of the metal binding site of bleomycin A2. The Cu(II) and Zn(II) derivatives. 7 23

The 13C NMR spectra at 25.2 MHz of the Zn(II) and Cu(II) complexes of the antitumor antibiotic bleomycin A2 are discussed. Complexation of the drug to Zn(II) causes 38 of the 52 resonance lines of bleomycin A2 to shift to new positions. All but ten of these shifted lines have been assigned in the Zn(II) bleomycin complex. Although the specific donor sites of the drug cannot be identified from the 13C NMR data, the analysis clearly shows that the pyrimidine-imidazole portion of the molecule is affected by chelation. This finding is in agreement with the previously reported metal-binding site of the antibiotic. The analysis also shows that carbon atoms which have large through-bond distances from the binding site can experience substantial chemical-shift changes upon metal binding. Complexation of the drug to Cu(II) eliminates 23 resonances from the spectrum of the molecule. All of these resonances emanate from carbon atoms which are located in the pyrimidine-imidazole portion of the drug.
 ...
PMID:Transition-metal binding site of bleomycin A2. A carbon-13 nuclear magnetic resonance study of the zinc(II) and copper(II) derivatives. 8 85

The reassignment of the 1H NMR C-2 histidine signals of the bovine pancreatic ribonuclease A has required a revision of the 1H NMR data on the role of the different histidines in their interaction with the Cu2+. The results of our measurements carried out at p2H 5.5 and 7.0 reduce the importance of His-12 as main site of interaction. At p2H 5.5 a very strong binding site involves His-119, while a weaker one contains certainly His-105. On the contrary, at p2H 7.0 the histidines 105 and 119 seem to possess binding constants of the same order of magnitude and in addition they provide stronger ligands for the Cu2+ than His-12. The comparison with X-ray data in the crystal shows numerous analogies. Finally, preliminary results on the competitive inhibition effect between the Cu2+ and 2',3'-cytidine monophosphoric acid are discussed.
 ...
PMID:1H NMR study on the interaction of cupric ion with ribonuclease A. 23 64

1. The proton NMR spectra of oxidised and reduced French bean plastocyanin have been recorded on a 270 MHz pulsed spctrometer. 2. The spectrum of a mixture containing the protein in the paramagnetic Cu(II) and diamagnetic Cu(I) states is a superposition of the separate spectra. When ferrirate spectra. 3. The results show that self-exchange between Cu(II)- and Cu(I)-plastocyanin is slow on the NMR time scale (kex less than 2-10(4) M-1-s-1 at 50 degrees C), and that electron transfer in the presence of ferricyanide is rapid (k greater than 1-10(5) M-1-s-1).
 ...
PMID:An NMR investigation of electron transfer in the copper-protein, plastocyanin. 24 Apr 33

The copper(I) complexes of peptides containing sulfhydryl and imidazole groups have been investigated in aqueous solution by proton magnetic resonance (1H NMR). The N-mercaptoacetyl-L-histidine coordinates through the sulfhydryl sulfur and imidazole nitrogen atoms to form the unique 1:1 Cu(I) complex species containing bridged imidazole. The sulfhydryl and imidazole groups are adequate ligands for not only Cu(II) but also Cu(I). The 1H NMR spectra show that the peptide nitrogen group is not involved in complex formation of Cu(I) with N-mercaptoacetyl-L-histidine and 3-mercaptopropionyl-L-histidine. The results have been discussed with respect to the protein ligands for 'blue' copper centers.
 ...
PMID:Proton-magnetic-resonance study of copper(I) complexes with peptides containing sulfhydryl and imidazole groups as possible model ligands for copper proteins. 91 6

Two modified forms of heparin, polymers A and B, have been prepared, one containing residues of nonsulfated alpha-L-idopyranosyluronic acid (3) and the other residues of alpha-L-galactopyranosyluronic acid (7), in place of the normal alpha-L-idopyranosyluronic acid 2-sulfate (1). In addition, both A and B contained 2-acetamido-2-deoxy-alpha-D-glucopyranosyl 6-sulfate residues (6) in place of the corresponding N-sulfated residues (2) of the original heparin. These polymers were subjected to sulfation under various conditions. Examination of the products by NMR spectroscopy showed that polymer A was sulfated initially at position-3 of residue 3, and that slower substitution occurred at position-3 of 6. By contrast, polymer B exhibited low regioselectivity, as sulfation occurred with about equal facility at positions-2 and -3 of 7, and -3 of 6. The sulfation products had no significant anti Xa activity. Based on the paramagnetic effects of Cu2+ and chemical shift displacements induced by Ca2+, NMR spectroscopy was used to compare cation-binding properties of A and B with those of heparin. In contrast to heparin, which forms a complex with Cu2+ detectable at a level of < 10(-3) mol per dimeric unit of the polymer, neither A nor B exhibited an interaction with the cation. However, polymer A was found to bind Ca2+, in this respect being distinct from the related modification, 1-->6, which contains a 2-sulfate group in 1, as well as from polymer B.
 ...
PMID:Regioselectivity in the sulfation of some chemically-modified heparins, and observations on their cation-binding characteristics. 129 Oct 46

The R-state conformation of the Cu(II)-substituted insulin hexamer has been identified, and a number of its derivatives have been studied via 1H NMR, ESR, and UV-visible spectroscopy. This work establishes that the Cu(II)-substituted insulin hexamer undergoes an analogous T to R conformational transition in solution that has been identified previously for Zn(II)- and Co(II)-insulin hexamers [Roy, M., Brader, M.L., Lee, R. W.-K., Kaarsholm, N.C., Hansen, J., & Dunn, M.F. (1989) J. Biol. Chem. 264, 19081-19085]. The data indicate that each Cu(II) center of the R-state Cu(II)-insulin hexamer possesses a coordination site that is accessible to anions from solution. Both phenol and anionic ligands that coordinate to the Cu(II) ions are required to generate the necessary heterotropic interactions that stabilize the R-state structure. With phenylmethylthiolate (PMT), a Cu(II)-R6 adduct that displays the spectral features of blue (type 1) copper proteins is obtained. This complex is proposed to embody a pseudotetrahedral CuIIN3S(PMT) chromophore, in which N is HisB10 (imidazolyl). The remaining ligands examined gave rise to Cu(II)-R6 adducts that possessed the spectral characteristics of normal (type 2) Cu(II) proteins. Under reducing conditions, Cu(I)-T6 and Cu(I)-R6 hexamers have been identified.
 ...
PMID:The T to R transition in the copper(II)-substituted insulin hexamer. Anion complexes of the R-state species exhibiting type 1 and type 2 spectral characteristics. 131 58

To study the importance of a rigid copper site for the structure and function of azurin, a mutant with a reduced number of internal hydrogen bonds around the copper has been prepared and characterized. To this purpose, the previously cloned azu gene from Alcaligenes denitrificans (Hoitink, C. W. G., Woudt, L. P., Turenhout, J. C. M., Van de Kamp, M., and Canters, G. W. (1990) Gene (Amst.) 90, 15-20) was expressed in Escherichia coli and an isolation and purification procedure for the azurin was developed. The azurin obtained after heterologous expression in E. coli appears spectroscopically indistinguishable from azurin derived from A. denitrificans. The hydrogen bonding network around the copper site was altered by replacing Asn47 by a leucine by means of site-directed mutagenesis. Asn47 is a conserved residue in all blue copper proteins of which the primary structure has been reported. Characterization of the mutant protein with UV-visible, electron spin resonance, and NMR spectroscopy, and comparison with the wild type azurin revealed that the structure of the copper site as well as the overall structure of the protein have been largely retained. The redox activity as measured by the electron self-exchange rate appears not to have changed either. However, the mutant differs from the wild type azurin with respect to stability and midpoint potential. Midpoint potentials of mutant and wild type azurin amount to 396 and 286 mV, respectively. The difference is due to sizable entropic and enthalpic contributions which to a large extent cancel. Possible explanations for the outcome of these experiments are discussed.
 ...
PMID:The importance of Asn47 for structure and reactivity of azurin from Alcaligenes denitrificans as studied by site-directed mutagenesis and spectroscopy. 132 Nov 27

Proton ENDOR has been observed from frozen solutions (ca. 38K degrees) of copper meso-(4-N-tetra-methylpyridyl)porphyrin (CuTMpyP(4)) complexed with Salmon sperm DNA in water and D2O. Lines from exchangeable protons of the DNA bases have been observed in these ENDOR spectra. Analyses of these ENDOR data show that the separations of these DNA protons from the copper atom are between 3.76 and 3.84 A with angles of 19.5 to 22.5 degrees between the Cu-H vectors and the gz axis. A distant ENDOR response has also been observed from phosphorous nuclei in the DNA backbone. We estimate that the phosphorous atoms producing this ENDOR signal are 7.5-10 A from the copper center of the porphyrin. These ENDOR data combined with results from an earlier NMR investigation have been used to construct a computer simulated model of the binding site in which the porphyrin is partially intercalated and extends into the major groove of DNA. The two GC base pairs at this site are slightly inequivalent. For each, the G imino proton and one of the C amino protons are at appropriate positions to account for the ENDOR signals arising from exchangeable protons. It is unlikely that this inequivalence would persist at room temperature where dynamic processes would give an apparently symmetric interaction. Although the model accounts for all reported experimental data involving tetracationic porphyrin species which have been suggested to be intercalators, it is not a unique solution.
 ...
PMID:Model for the porphyrin-DNA binding site: ENDOR investigations of Cu-porphyrins binding to DNA. 132 80


1 2 3 4 5 6 7 8 9 10 Next >>