PubMed Health⌕ Search

Biomedical subjects

F Inagaki

Publications and source records attributed to F Inagaki.

At least 145 records · Page 8Linked to original sources
PubMed1982

Structural dynamics of erabutoxin b. A 13C nuclear magnetic resonance relaxation study of methyl groups.

A detailed examination has been made of the 13C NMR relaxation times of the assigned methyl groups of erabutoxin b. Anisotropic rotation was analysed using a restricted diffusion model. The results are compared with a previous study of the 1H NMR relation times. There is good agreement on the segments of the molecule which show restricted motion. Our results are compared with those of previous studies on proteins in solution and in crystals and again the general agreement is good. We have attempted to interpret the value of the limited motion seen in the protein to the reactions of the neurotoxin.

Elapid Venoms↗
PubMed1982

Local conformational transition of toxin B from Naja naja as studied by nuclear magnetic resonance and circular dichroism.

The 270-MHz proton NMR spectra of toxin B, a long neurotoxin from Naja naja, in 2H2O solution were analyzed. From the analysis of the discontinuous pH dependence of His-21 C-2 proton chemical shift a 23 degrees C, toxin B is found to exhibit a slow-exchange conformational transition between the states A and B with the transition midpoint at pH 5.75. Separate peaks due to the states A and B were also observed for His-21 C-4 proton, Tyr-25 ring protons, phenylalanine ring protons and two C-alpha protons. This local conformational transition as induced by the protonation of His-21 residue is further confirmed by the measurement of the pH dependence of circular dichroism. In state A, the pKa value of His-21 is 6.6-6.9, indicating that His-21 residue is not significantly perturbed. The His-21 residue in state B is accessible to solvent, as found by the analysis of hydrogen leads to deuterium exchange rate. The low pKa value (4.6-4.9) of His-21 in stat B is probably due to the formation of a hydrogen bond of the imidazole ring. The conformational transition rate at 23 degrees C is obtained as 2.5 s-1 at the transition midpoint (pH 5.75), by the saturation transfer method. The conformational transition at 60 degrees C corresponds to a fast exchange case for the Tyr-25 ring proton resonances and to an intermediate exchange case for the His-21 C-2 and C-4 proton resonances. The transition rate at the midpoint is about 300 s-1 at 60 degrees C.

Circular Dichroism↗
PubMed1982

Dynamics of erabutoxin b as studied by nuclear magnetic resonance. Relaxation studies of methyl proton resonances.

Longitudinal and transverse relaxation times were measured for well-resolved and assigned methyl proton resonances of erabutoxin b at 270 MHz, 300 MHz and 500 MHz. Both longitudinal and transverse magnetization decay curves are non-exponential due to cross-relaxation and cross-correlation effects. The longitudinal and transverse relaxation rates were obtained from the initial slope of both magnetization decay curves. The correlation times for the isotropic tumbling motion of the protein were determined to be 2.82 ns at 300 K and 1.62 ns at 330 K from the analysis of the relaxation data of some alpha protons. Using these values, the relaxation data of methyl protons were fitted to various theoretical models. Most of the methyl resonances could be fitted well to a model which allowed methyl rotation (in the range 0.01-0.05 ns) and an external contribution from protons assumed to be in positions derived from X-ray coordinates. The data for a few methyl groups, however, could not be fitted in this way. For these a smaller number of external protons than predicted by the X-ray coordinates was assumed. Additionally, a larger amplitude motion had to be introduced into the model for particular residues. This additional motion requires concerted protein motion close to these residues, since the X-ray structure suggests that steric hindrance would prevent local motion. These results are consistent with the idea of a flexible and dynamic structure for proteins.

Elapid Venoms↗
PubMed1982

Individual assignments of the amide proton resonances involved in the triple-stranded antiparallel pleated beta-sheet structure of a long neurotoxin, Laticauda semifasciata III from Laticauda semifasciata.

THe characteristic feature of the crystal structure of erabutoxin b, a short neurotoxin from Laticauda semifasciata, and alpha-cobratoxin, a long neurotoxin from Naja naja siamensis, is the presence of a triple-stranded antiparallel pleated beta-sheet structure formed by the central and the third peptide loops. In the present study, we have studied the assignment of slowly exchangeable amide protons of Laticauda semifasciata III from L. semifasciata, using nuclear Overhauser effects (NOE) and spin-decoupling methods. The results show that nearly all of the slowly exchangeable amide protons are to be assigned to the back-bone amide protons, involved in the triple-stranded antiparallel pleated beta-sheet structure, indicating that this sheet is stable in 2H2O solution. In contrast, the amide protons in short neurotoxins are readily exchangeable under the same experimental condition, suggesting that long neurotoxins have a more rigid sheet structure than short ones. This rigidity may come from the hydrophobic and hydrogen bond interaction between the central loop and the tail, which is not present in short neurotoxins. Since the functionally important residues are located on this beta-sheet, the different kinetic properties of the neurotoxins are well correlated with the difference in the rigidity of the beta-sheet.

Amines↗
PubMed1982

Two-dimensional NMR spectroscopy of siomycin A. Proton--carbon-13 chemical shift correlation.

A trial application of a recent two-dimensional nuclear magnetic resonance experiment to the polypeptide antibiotic siomycin A is described. Proton--carbon-13 chemical shift correlation measures the proton and carbon-13 chemical shift for each directly bonded CH group in a molecule, in a single experiment. The resultant map of correlated chemical shifts enables the carbon-13 spectrum to be assigned directly from the known proton shifts, and allows individual proton signals to be identified without problems of overlap. The signal-to-noise ratio available from such techniques should enable their application to aqueous protein solutions using currently available high-field spectrometers.

Anti-Bacterial Agents↗
PubMed1981

Structural differences between erabutoxins in aqueous solution and in crystalline states.

The snake venom toxins erabutoxin a and b, have been studied in detail in aqueous solution over a wide pH range by proton NMR spectroscopy. Great care has been taken particularly to assign and locate in the structure residue histidine-7 with reference to other amino-acid sidechains. It lies in a pocket away from the surface in agreement with its lack of chemical reactivity. The data are not consistent with two structure determinations in crystals by X-ray diffraction. The aqueous solution conformation explains in greater depth the observed essentially of amino acids to the activity of the toxin.

Crystallization↗
PubMed1981

Advanced nuclear magnetic resonance lanthanide probe analyses of short-range conformational interrelations controlling ribonucleic acid structures.

An advanced method was developed for lanthanide-probe analyses of the conformations of flexible biomolecules such as nucleotides. The new method is to determine structure parameters (such as internal-rotation angles) and population parameters for local conformational equilibria of flexible sites, together with standard deviations of these parameters. As the prominent advantage of this method, the interrelations among local conformations of flexible sites may be quantitatively elucidated from the experimental data of lanthanide-induced shifts and relaxations and vicinal coupling constants. As a structural unit of ribonucleic acids, the molecular conformations and conformational equilibria of uridine 3'-monophosphate in aqueous solution were analyzed. The stable local conformers about the C3'-O3' bond are the G+ (phi' = 281 +/- 11 degrees) and G- (phi' = 211 +/- 8 degrees) forms. The internal rotation about the C3'-O3' bond and the ribose-ring puckering are interrelated; 97 +/- 5% of the C3'-endo ribose ring is associated with the G- form while 70 +/- 22% o the C2'-endo ribose ring is associated with the G+ form. An interdependency also exists between the internal rotation about the C4'-C5' bond and the ribose-ring puckering. These short-range conformational interrelations are probably important in controlling the dynamic aspects of ribonucleic acid structures.

Chemical Phenomena↗
PubMed1981

Proton-nuclear-magnetic-resonance study on molecular conformations of long neurotoxins. alpha-Bungarotoxin from Bungarus multicinctus and Toxin B from Naja naja.

The 270-MHz proton NMR spectra were analyzed of the long neurotoxins alpha-bungarotoxin from Bungarus multicinctus and Toxin B from Naja naja. The aromatic proton resonances were completely assigned to individual nuclei for alpha-bungarotoxin and in part for toxin B. The pH dependences of proton chemical shifts were analyzed by the nonlinear least-square method, for obtaining pKa values and protonation shifts. The pKa values of Tyr-25, an invariant residue of neurotoxins, are 12.1 for alpha-bungarotoxin and 11.3 for toxin B, suggesting the presence of a strong hydrogen bond involving Tyr-25 in alpha-bungarotoxin. The Trp-29 residues of both toxins show a common titration shift due to the carboxylate group of Asp-31 and a similar structural arrangement of functionally invariant pair of Trp-29 and Asp-31 is implied. From the temperature dependences of the chemical shifts of His-68 and a methyl group of alpha-bungarotoxin, the local structure around His-68 near the tail part is shown to be more flexible than the other part. The six main-chain amide protons of alpha-bungarotoxin exchange most slowly with solvent deuterons and are found by interproton nuclear Overhauser effects to be in the beta-sheet near the aromatic ring of Tyr-25 residue. Hydrogen leads to deuterium exchange rates in 2H2O solution at 37 degrees C were measured of slowly exchanging amide protons of alpha-bungarotoxin, toxin B, and two short neurotoxins, namely cobrotoxin and erabutoxin b. The two long neurotoxins have amide protons with relatively long half-times spanning as long as 10-100 h, but the two short neurotoxins do not have amide protons with half-times longer than 3 h. The distributions of the half-times of amide proton exchange indicate the structural rigidity of neurotoxins in the order, alpha-bungarotoxin greater than toxin B greater than cobrotoxin approximately erabutoxin b, in agreement with the order of neurotoxicity as reported previously by Chicheportiche et al. and by Lee and Chen.

Amino Acid Sequence↗
PubMed1981

A proton-magnetic-resonance study on the molecular conformation and structure-function relationship of a long neurotoxin, laticauda semifasciata III from Laticauda semifasciata.

The 300-MHz and 500-MHz NMR spectra of a long neurotoxin laticauda semifasciata III (LS III) from Laticauda semifasciata have ben analysed. Comparison with the NMR spectra of alpha-cobratoxin from Naja naja siamensis, a homologous long neurotoxin to laticauda semifasciata III, allowed the assignment of all the aromatic protein resonances to specific amino acid residues. All the methyl proton resonances have been assigned to specific types of amino acid residues. The pH dependences of the aromatic and methyl proton chemical shifts were analyzed by the non-linear least-square method to give the pKa values and protonation shifts. The interproton nuclear Overhauser effect enhancements were measured in order to elucidate the spatial proximity of the methyl-bearing residues and aromatic residues. On the basis of these NMR data and using the crystal structure of alpha-cobratoxin by Walkinshaw et al., more than half of the methyl proton resonances have been assigned to specific amino acid residues. A hydrophobic core comprising the first loop, the central loop and the tail part of the molecule has been defined. This hydrophobic core may be common to all long neurotoxins and may protect the three-stranded antiparallel pleated beta-sheet structure, thus making the backbone structure of long neurotoxins more rigid than that of short neurotoxins. The positively charged surface of laticauda semifasciata III, which is responsible for binding to the acetylcholine receptor protein, is confirmed as the concave surface formed by the central and the third loop. The arrangement of the amino acid residues on this surface is similar to that of all other neurotoxins. Accordingly, the slow on-off rates of association of long neurotoxins with receptor is considered to arise from the rigid backbone structure. A small conformation change is thought to be associated with binding to the receptor protein.

Amino Acid Sequence↗
PubMed1981

Nuclear magnetic resonance study on the microenvironments of histidine residues of ribonuclease T1 and carboxymethylated ribonuclease T1.

The 270-MHz 1H NMR spectra and fluorescence of ribonuclease T1 and carboxymethylated ribonuclease T1 were measured in aqueous solution. Histidine C4 proton resonances were assigned to individual residues. From the pH dependences of the chemical shifts of histidine C2 and C4 protons, the pKa values of histidine residues were obtained by the non-linear least-squares method. The hydrogen leads to deuterium exchange rates of histidine C2 protons were determined as a measure of the accessibility of histidine residues to the solvent. Each histidine residue of ribonuclease T1 was found to interact with a carboxylate group of an aspartic or glutamic acid residue; in particular, His-40 was shown to interact with Glu-58. Upon carboxymethylation of Glu-58, His-92 and His-27 are more shielded from the solvent while His-40 remains exposed to the solvent. The 67.9-MHz 13C NMR spectra were measured for the 13C-enriched preparation of carboxymethylated ribonuclease T1. From the pH dependence of 13C chemical shift, the pKa value of the carboxymethylated Glu-58 was found to be unusually low, suggesting the formation of an ionic or hydrogen bond between this carboxymethyl group and a positively charged group, possibly of Arg-77.

Chemical Phenomena↗
PubMed1980

Molecular conformation and function of erabutoxins as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of erabutoxins a, b and c from Laticauda semifasciata in 2H2O solution were observed together with [15-N6-acetyllysine]erabutoxin b, [27-N6-acetyllysine]-erabutoxin b and [47-N6-acetyllysine]erabutoxin b. The lysine epsilon-methylene proton resonances of erabutoxin b are assigned to individual residues. The epsilon-methylene proton resonance of Lys-27 is significantly broad, indicating that the mobility of this residue is restricted. Upon acetylation of Lys-27 of erabutoxin b, the pKa values of three other lysine residues are lowered by about 0.2, indicating long-range interactions among lysine residues. All the methyl proton resonances are assigned to amino acid types, primarily by the spin-echo double-resonance method. The pH dependences of proton chemical shifts were analyzed by the nonlinear least-square method, for obtaining pKa values and protonation shifts. The interproton nuclear Overhauser effect enhancements were measured for elucidating the spatial proximity of methyl-bearing residues and aromatic residues. On the basis of these NMR data and with the crystal structures by Low et al. and by Petsko et al., the methyl proton resonances of all the valine, leucine, and isoleucine residues and Thr-45 have been identified. The microenvironments of Tyr-25, His-26, Trp-29, four lysines and eight methyl-bearing residues have been elucidated. The addition of the paramagnetic hexacyanochromate ion causes broadening of the proton resonances of Thr-45, Lys-47, Ile-50, Trp-29 and Ile-36 residues located on one end of the molecule of erabutoxin b. The positively charged invariant residues of Lys-47 and Arg-33 at this part of the molecule are probably involved in the binding to the receptor protein.

Chromates↗