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1.
The most plausible set of chemical shift assignments for alpha-bungarotoxin as deduced from the combined use of two-dimensional J-correlated and two-dimensional nuclear Overhauser effect 1H nuclear magnetic resonance (NMR) spectroscopy was in conflict with the accepted amino acid sequence between residues 8 and 12 and residues 66 and 70 [Basus, V. J., Billeter, M., Love, R. A., Stroud, R. M., & Kuntz, I. D. (1988) Biochemistry (first paper of three in this issue]). Furthermore, NMR spectra of alpha-bungarotoxin, purified by conventional methods, evidenced a second species at the level of approximately 10% total protein. The minor component was separated from alpha-bungarotoxin by Mono-S (cationic) chromatography. Sequencing of Mono-S-purified alpha-bungarotoxin and one of its tryptic peptides showed that the correct sequence for alpha-bungarotoxin is Ser-Pro-Ile at positions 9-11 and Pro-His-Pro at positions 67-69. The electron density map of alpha-bungarotoxin [Love, R. A., & Stroud, R. M. (1986) Protein Eng. 1, 37] was refined with the new sequence data. Improvements in the structure were found primarily for residues 9-11. Sequence analysis of two overlapping tryptic peptides proved that the minor species differed from alpha-bungarotoxin by replacement of a valine for an alanine at position 31. This new toxin, alpha-bungarotoxin(Val-31), binds to the acetylcholine receptor with an affinity that is comparable to that of alpha-bungarotoxin. 相似文献
2.
Jonathan H. Davis David A. Agard Tracy M. Handel Vladimir J. Basus 《Journal of biomolecular NMR》1997,10(1):21-27
-Lytic protease, a bacterial serine protease of 198 aminoacids (19800 Da), has been used as a model system for studies of catalyticmechanism, structure–function relationships, and more recently forstudies of pro region-assisted protein folding. We have assigned thebackbones of the enzyme alone, and of its complex with the tetrahedraltransition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boroVal, usingdouble- and triple-resonance 3D NMR spectroscopy on uniformly15N- and 13C/15N-labeled protein.Changes in backbone chemical shifts between the uncomplexed and inhibitedform of the protein are correlated with distance from the inhibitor, thedisplacement of backbone nitrogens, and change in hydrogen bond strengthupon inhibitor binding (derived from previously solved crystal structures).A comparison of the solution secondary structure of the uninhibited enzymewith that of the X-ray structure reveals no significant differences.Significant line broadening, indicating intermediate chemical exchange, wasobserved in many of the active site amides (including three broadened toinvisibility), and in a majority of cases the broadening was reversed uponaddition of the inhibitor. Implications and possible mechanisms of this linebroadening are discussed. 相似文献
3.
Two-dimensional 1H NMR of three spin-labeled derivatives of bovine pancreatic trypsin inhibitor 总被引:2,自引:0,他引:2
P A Kosen R M Scheek H Naderi V J Basus S Manogaran P G Schmidt N J Oppenheimer I D Kuntz 《Biochemistry》1986,25(9):2356-2364
Three nitroxide spin-labeled monoderivatives of bovine pancreatic trypsin inhibitor were prepared with the amino-specific reagent succinimidyl 1-oxy-2,2,5,5-tetramethyl-3-pyrroline-3-carboxylate. The monoderivatives were purified by ion-exchange and affinity chromatography. Thin-layer maps of tryptic peptides of the monoderivatives showed that the spin-label was incorporated at either the alpha-amino group, Lys-15, or Lys-26. Two-dimensional J-correlated 1H NMR spectra of the monoderivatives were recorded. Spectra were also recorded after reduction by ascorbic acid of the nitroxide label to hydroxylamine. With the nitroxide label present, significant line-broadening effects on many of the cross peaks in the spectra were observed. The extent of line broadening for the C alpha H-NH cross peaks was qualitatively correlated with the distance between the labeled amino group and the average C alpha H-NH position in the crystal structure. The spin-label affects cross peaks of protons within approximately 15 A. This study suggests that it is feasible to accumulate sufficient intramolecular distances in order to determine protein solution structures with the aid of distance geometry algorithms. 相似文献
4.
15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferrocytochrome b5 have been obtained, using 15N-1H heteronuclear correlation methods employing globally 15N-labeled protein. Unlike other cytochrome b5 species assigned to date (Guiles et al., 1990) the rat cytochrome exists as an equilibrium distribution of conformers in nearly equal abundance (Lee et al., 1990). The ratio of conformers present in all other species variants is approximately 1:9. More than 40% of all residues of the rat protein exhibit NMR-detectable heterogeneity due to the 180 degrees rotation of the heme about the alpha, gamma-meso axis. NOESY and HOHAHA relayed 15N-1H double-DEPT heteronuclear correlation methods were an indispensible tool for the deconvolution of a system with this level of heterogeneity. Differences in the resonance assignments between the two equilibrium conformers were found to be as great as differences between species variants we have previously reported. On the basis of the magnitude and extent of the observed chemical shift differences and specific NOESY connectivities observed in the two isomers, we believe the two equilibrium conformers differ not only by a simple back-to-front flip of the heme but also by an additional rotation about an axis normal to the heme plane as has been previously suggested by Pochapsky et al. (1990). A short segment of the protein at the N-terminus could not be assigned, presumably due to rapid exchange of solvent-accessible amide protons in this disordered segment of the protein. Assignments for 93 of the 98 residues of this 12-kDa protein have been obtained. 相似文献
5.
Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments 总被引:2,自引:0,他引:2
We report the complete sequence-specific assignment of the backbone resonances and most of the side-chain resonances in the 1H NMR spectrum of alpha-bungarotoxin by two-dimensional NMR. Problems with resonance overlap were resolved with the assistance of the HRNOESY experiment described in an accompanying paper [Basus, V.J., & Scheek, R.M. (1988) Biochemistry (second paper of three in this issue)]. Significant differences exist between the solution structure described here and the crystal structure of alpha-bungarotoxin, on the basis of the proton to proton distances obtained by nuclear Overhauser enhancement spectroscopy (NOESY) and the corresponding distances from the X-ray crystal structure [Love, R.A., & Stroud, R.M. (1986) Protein Eng. 1, 37]. These differences include a larger beta-sheet in solution and a different orientation of the invariant tryptophan, Trp-28, making the solution structure more consistent with the crystal structure of the homologous neurotoxin alpha-cobratoxin. Four errors in the order of the amino acids in the primary sequence were indicated by the NMR data. These errors were confirmed by chemical means, as described in an accompanying paper [Kosen, P.A., Finer-Moore, J., McCarthy, M.P., & Basus, V.J. (1988) Biochemistry (third paper of three in this issue)]. 相似文献
6.
Cytochrome c associated apoptosis during ATP recovery after hypoxia in neonatal rat cerebrocortical slices 总被引:2,自引:0,他引:2
Cellular injury was evaluated in superfused cerebrocortical slices (350 micro m) from 7-day-old Sprague-Dawley rats exposed to 30 min hypoxia followed by 4 h of reoxygenation. At the end of hypoxia homogenous cytosolic immunoreactivity of cytochrome c increased approximately fourfold, cytochrome c intensity in western blot analyses increased more than fivefold, and whole cell and cytosolic cleaved caspase-9 underwent 50% and 100% increases, respectively. Immunostaining of sections taken 1.5 h after hypoxia showed: (i) more than a threefold increase in cleaved caspase-9; (ii) localization of cleaved caspase-9 to the interior and peripheral exterior of nuclei; and (iii) homogeneously distributed cytochrome c in the cytosol. Western blot analysis for 1.5 h after hypoxia showed that cytosolic caspase-9 returned to control values, while whole cell caspase-9 stayed approximately the same, suggesting translocation of caspase-9 to nuclei. By 4 h after hypoxia there was significant nuclear fragmentation and an increase in TUNEL positive staining. 31P/1H nuclear magnetic resonance (NMR) confirmed substantial decreases of ATP and phosphocreatine during hypoxia, with rapid but incomplete recovery being close to steady state 1 h after reoxygenation. At all time points after hypoxia the primary injury was cytochrome c associated apoptosis. 相似文献
7.
Martin D Shetlar Vladimir J Basus Arnold M Falick Anwer Mujeeb 《Photochemical & photobiological sciences》2004,3(10):968-979
The photochemical reactions of 5-methylcytosine (m(5)C), a minor component of mammalian DNA, have been studied at a concentration of 2 mM in frozen 10 mM aqueous NaCl solution at dry ice temperature (194.5 K). For these studies, low-pressure lamps emitting mainly UVB radiation were used. We have isolated and characterized three cyclobutane dimers, namely the cis-anti(c,a) the cis-syn(c,s) and the trans-syn(t,s) forms. While the c,a and the t,s cyclobutane dimers are relatively stable towards deamination upon standing in solution at 277 K, the c,s isomer is gradually converted into the corresponding c,s m(5)C-thymine (Thy) mixed dimer; this latter reaction occurs considerably faster at 310 K. The t,s cyclobutane dimer is converted into the corresponding m(5)C-Thy mixed dimer upon incubation at 373 K, while the c,a dimer is converted into a mixture of m(5)C and c,a mixed dimer when incubated at 310 K. Irradiation of equimolar mixtures of Thy (1 mM) and m(5)C (1 mM) under similar conditions yields each of the three m(5)C cyclobutane dimers, as well as significant amounts of c,a, c,s and t,s m(5)C-Thy mixed cyclobutane dimers. These m(5)C-Thy dimers undergo decompositions similar in nature to the processes undergone by m(5)C cyclobutane dimers. Pseudo-first order rate constants for deamination of the c,s m(5)C homodimer and c,s m(5)C-Thy heterodimer at various temperatures and at pH 7.7 have been measured and the enthalpies and entropies of activation have been evaluated for the deamination processes for these two compounds. The two dimers have half-lives of about 14 and 22 h, respectively, at 310 K; however, at 273 K, the corresponding half-lives can be evaluated as being around 30 and 36 days, respectively. 相似文献
8.
Complete sequence-specific assignments of the 1H NMR spectrum of bungarotoxin were reported in the previous paper [Basus, V.J., Billeter, M., Love, R.A., Stroud, R.M., & Kuntz, I.D. (1988) Biochemistry (first paper of three in this issue)]. The assignment was significantly aided by the use of the homonuclear Hartman-Hahn relayed coherence transfer nuclear Overhauser enhancement spectroscopy experiment (HRNOESY) which we present here, as a modification of relayed coherence transfer nuclear Overhauser enhancement spectroscopy (relayed NOESY) [Wagner, G. (1984) J. Magn. Reson. 57, 497]. As shown here, HRNOESY resolves problems of proton resonance overlap especially in extended chain conformations as found in beta-sheets. 相似文献
9.
Catechol 2, 3-dioxygenase is present in several types of bacteria and undergoes degradation of environmental pollutants through
an important key biochemical pathways. Specifically, this enzyme cleaves aromatic rings of several environmental pollutants such
as toluene, xylene, naphthalene and biphenyl derivatives. Hence, the importance of Catechol 2, 3-dioxygenase and its role in the
degradation of environmental pollutants made us to predict the three-dimensional structure of Catechol 2, 3-dioxygenase from
Burkholderia cepacia. The 10ns molecular dynamics simulation was carried out to check the stability of the modeled Catechol 2, 3-
dioxygenase. The results show that the model was energetically stable, and it attains their equilibrium within 2000 ps of production
MD run. The docking of various petroleum hydrocarbons into the Catechol 2,3-dioxygenase reveals that the benzene, O-xylene,
Toluene, Fluorene, Naphthalene, Carbazol, Pyrene, Dibenzothiophene, Anthracene, Phenanthrene, Biphenyl makes strong
hydrogen bond and Van der waals interaction with the active site residues of H150, L152, W198, H206, H220, H252, I254, T255,
Y261, E271, L276 and F309. Free energy of binding and estimated inhibition constant of these compounds demonstrates that they
are energetically stable in their binding cavity. Chrysene shows positive energy of binding in the active site atom of Fe. Except
Pyrene all the substrates made close contact with Fe atom by the distance ranges from 1.67 to 2.43 Å. In addition to that, the above
mentioned substrate except pyrene all other made π-π stacking interaction with H252 by the distance ranges from 3.40 to 3.90 Å.
All these docking results reveal that, except Chrysene all other substrate has good free energy of binding to hold enough in the
active site and makes strong VdW interaction with Catechol-2,3-dioxygenase. These results suggest that, the enzyme is capable of
catalyzing the above-mentioned substrate. 相似文献
10.
Leonard Moise Andrea Piserchio Vladimir J Basus Edward Hawrot 《The Journal of biological chemistry》2002,277(14):12406-12417
We report a new, higher resolution NMR structure of alpha-bungarotoxin that defines the structure-determining disulfide core and beta-sheet regions. We further report the NMR structure of the stoichiometric complex formed between alpha-bungarotoxin and a recombinantly expressed 19-mer peptide ((178)IPGKRTESFYECCKEPYPD(196)) derived from the alpha7 subunit of the chick neuronal nicotinic acetylcholine receptor. A comparison of these two structures reveals binding-induced stabilization of the flexible tip of finger II in alpha-bungarotoxin. The conformational rearrangements in the toxin create an extensive binding surface involving both sides of the alpha7 19-mer hairpin-like structure. At the contact zone, Ala(7), Ser(9), and Ile(11) in finger I and Arg(36), Lys(38), Val(39), and Val(40) in finger II of alpha-bungarotoxin interface with Phe(186), Tyr(187), Glu(188), and Tyr(194) in the alpha7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the alpha7 receptor, places alpha-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site. The disulfide-rich core of alpha-bungarotoxin is suggested to be tilted in the direction of the membrane surface with finger II extending into the proposed ligand-binding cavity. 相似文献