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Biomedical subjects

D Kohda

Publications and source records attributed to D Kohda.

47 records · Page 3Linked to original sources
PubMed1990

Nonprotein amino acid furanomycin, unlike isoleucine in chemical structure, is charged to isoleucine tRNA by isoleucyl-tRNA synthetase and incorporated into protein.

Nonprotein amino acid furanomycin was found to bind with Escherichia coli isoleucyl-tRNA synthetase (IleRS) almost as tightly as the substrate L-isoleucine. The conformation of furanomycin bound to the enzyme was determined by NMR analyses including the transferred nuclear Overhauser effect method. The conformation of IleRS-bound furanomycin was similar to that of L-isoleucine, although the chemical structure of furanomycin is unlike that of L-isoleucine. By E. coli IleRS, E. coli tRNAIle was charged with furanomycin as efficiently as with L-isoleucine. Furthermore, furanomycyl-tRNAIle was bound to polypeptide chain elongation factor Tu as tightly as isoleucyl-tRNAIle. Furanomycin was found to be incorporated into beta-lactamase precursor by in vitro protein biosynthesis. A newly designed amino acid will probably be incorporated into proteins, provided that the new amino acid takes a similar conformation as a protein-constituting amino acid in the active site of an aminoacyl-tRNA synthetase.

Amino Acids↗
PubMed1989

Polypeptide chain fold of human transforming growth factor alpha analogous to those of mouse and human epidermal growth factors as studied by two-dimensional 1H NMR.

The 1H NMR spectrum of human transforming growth factor alpha (TGF-alpha) was analyzed almost completely by the sequential assignment method using two-dimensional NMR techniques. On the basis of the nearly complete sequence-specific resonance assignment, secondary and tertiary structures of human TGF-alpha in solution (pH 4.9, 28 degrees C) were determined to satisfy the upper limits of proton-proton distances derived from nuclear Overhauser effect experiments. Although human TGF-alpha and mouse epidermal growth factor (EGF) share 27% homology in amino acid sequence, the backbone chain folds in the two growth factors are quite similar. The structure and function of TGF-alpha is well characterized by the "mitten model" previously proposed for mouse EGF. The gross shape of the TGF-alpha molecule resembles a mitten. TGF-alpha interacts with the receptor as a mitten would grasp an object. However, there is an appreciable structural difference between the two growth factors in the back of the mitten that is formed by the N-terminal polypeptide segment. This is consistent with the evidence that the backs of these molecules are not involved in the receptor binding.

Amino Acid Sequence↗
PubMed1988

Complete sequence-specific 1H nuclear magnetic resonance assignments for mouse epidermal growth factor.

The 1H NMR spectrum of the mouse epidermal growth factor (53 residues) was analyzed with the use of two-dimensional NMR techniques. All the observable 296 proton resonances were completely assigned in a sequential manner. For the spin system identification, two-dimensional homonuclear Hartmann-Hahn spectrum was useful, especially for arginine and proline residues. The easy spin system identification of these long-side-chain-bearing amino acid residues greatly facilitated the sequence-specific resonance assignment of the epidermal growth factor.

Amino Acids↗
PubMed1988

Tertiary structure of mouse epidermal growth factor determined by two-dimensional 1H NMR.

The tertiary structure of mouse epidermal growth factor (EGF) in solution (28 degrees C, pH 2.0) was studied by two-dimensional NMR spectroscopy. Proton-proton distance constraints derived from NOESY spectra were used to construct a mechanical molecular model of mouse EGF, which was subsequently checked by means of a preliminary distance geometry calculation. The chain-folds in the two structural domains of mouse EGF were very similar to those previously reported (Montelione et al. (1987) Proc. Natl. Acad. Sci. U.S. 84, 5226-5230). However, the relative orientations of the two domains were different. Because we could assign much more inter-domain NOEs, the relative orientations of the two domains were well determined in our model. The hollow between the two domains may function as a binding site for the EGF receptor.

Animals↗
PubMed1988

A comparative 1H NMR study of mouse alpha(1-53) and beta(2-53) epidermal growth factors.

The three-dimensional structure of the mouse epidermal growth factor (EGF) in solution was studied by comparison of the 1H NMR spectra of alpha EGF (1-53) and beta EGF (2-53, des-asparaginyl 1 form). Using pH dependence of chemical shifts and a two-dimensional difference spectrum, the effect of the N-terminal deletion was investigated based on the complete assignment of the proton resonances. The affected residues were all found to be located exactly in the triple-stranded, beta-sheet core in the N-terminal domain of the EGF molecule.

Amino Acids↗
PubMed1987

NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase.

The 400-MHz 1H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS. The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche+ form about the C alpha-C beta bond and the trans form about the C beta-C gamma 1 bond. The IleRS-bound L-valine takes the gauche- form about the C alpha-C beta bond. Thus, the conformation of IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Analysis of NMR spectra of sugar chains of glycolipids by multiple relayed COSY and 2D homonuclear Hartman-Hahn spectroscopy.

We applied multiple relayed COSY and 2D homonuclear Hartman-Hahn spectroscopy to globoside, a glycolipid purified from human red blood cells. The subspectra corresponding to individual sugar components were extracted even from overlapping proton resonances by taking the cross sections of 2D spectra parallel to the F2 axis at anomeric proton resonances, so that unambiguous assignments of sugar proton resonances were accomplished.

Galactosamine↗
PubMed1987

Functions of isolated domains of methionyl-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8.

Methionyl-tRNA synthetase (MetRS, 2 X 75 kDa) was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB8. The polypeptide chain of MetRS was cleaved by limited digestion with trypsin into four domains: T1 (29 kDa), T2 (23 kDa), T3 (14.5 kDa), and T4 (7.5 kDa), which were aligned in that order. MetRS was also cleaved into similar fragments with a variety of other proteases. Domains T1, T2, T3, and T4 were isolated by column chromatography. "Tandem domain" T1-T2 (56 kDa) is fully active in the aminoacylation of tRNA and is further cleaved with trypsin into domains T1 and T2. Domain T1 is the smallest aminoacylation unit so far reported. Domain T2 (enzymatically inactive) interacts with tRNAMetf, as found by UV-induced cross-linking. Isolated domain T3 forms a dimer and is responsible for the dimer assembly of two protomers in MetRS. Domain T4 is a flexible tail of MetRS. These domains, in particular T1 and T2, will be important for detailed structure analyses in relation to aminoacylation activity.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases from an extreme thermophile Thermus thermophilus HB8: protein structure and Zn2+ binding.

Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases have been purified from an extreme thermophile, Thermus thermophilus HB8. Valyl-tRNA and isoleucyl-tRNA synthetases are found to be monomer proteins (Mr 108000 and 129000, respectively), while methionyl-tRNA synthetase is a dimer protein (Mr 150000). These enzymes are very similar with respect to amino acid compositions and alpha-helix contents as estimated by circular dichroism analyses. Furthermore, two Zn2+ are tightly bound to each of these synthetases. These data suggest that valyl-tRNA and isoleucyl-tRNA synthetases consist of two domains, each corresponding to the subunit of methionyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Aminoacyl-tRNA synthetases from an extreme thermophile, Thermus thermophilus HB8.

Thermostable aminoacyl-tRNA synthetases specific to Val, Ile, Met and Glu were purified from an extreme thermophile, Thermus thermophilus HB8. As for the subunit compositions and molecular weights, these four aminoacyl-tRNA synthetases are similar to the corresponding enzymes from E. coli and B. stearothermophilus. Val-tRNA, Ile-tRNA and Met-tRNA synthetases from T. thermophilus have two tightly bound zinc ions, whereas Glu-tRNA synthetase does not. The amino acid compositions and secondary structures of Val-tRNA, Ile-tRNA and Met-tRNA synthetases are quite similar to one another. The conformational transition involving the anticodon of E. coli tRNAGlu as complexed with Glu-tRNA synthetase from T. thermophilus is necessary for the aminoacylation activity.

Amino Acids↗