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T Hakoshima

Publications and source records attributed to T Hakoshima.

66 records · Page 4Linked to original sources
PubMed1988

Crystallization and preliminary X-ray investigation reveals that tumor necrosis factor is a compact trimer furnished with 3-fold symmetry.

Crystals of recombinant human tumor necrosis factor produced by Escherichia coli have been obtained under different conditions. Crystals suitable for X-ray studies are produced by a vapor diffusion technique using sodium phosphate as both precipitant and buffer at pH 6.5. The crystals belong to the cubic space group, P2(1)3 with unit cell dimensions a = b = c = 95.7 A (1 A = 0.1 nm). Preliminary photography reveals that the crystals are moderately stable to X-rays and diffract to at least 3 A resolution. The diffraction data for native crystals have been collected on a diffractometer at 3 A resolution. Another crystal form, which appeared in a solution containing sodium phosphate at pH 8.0, has the trigonal space group P3 with unit cell dimensions a = b = 63.8 A and c = 54.4 A, and produces measurable reflections to a resolution of 3 A. Hexagonal crystals also have been obtained by the use of polyethylene glycol as precipitant in the range pH 7.6 to 8.0; however, the crystals are fragile and unstable to X-rays. Conservation of 3-fold symmetry in the different crystal forms obtained could reflect the ability of tumor necrosis factor molecules to form trimers in solution and probably the nature of binding of the molecules to cellular receptors.

Cloning, Molecular↗
PubMed1988

Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45.

The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild-type enzyme are described. The gene for the mutant RNase T1 (Tyr45----Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene [Ikehara, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 4695-4699]. In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N- and C-termini. After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild-type RNase T1. The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased stacking effect in the binding pocket for the guanine base. This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild-type RNase T1.

Base Sequence↗
PubMed1988

Glu 46 of ribonuclease T1 is an essential residue for the recognition of guanine base.

The Glu 46 of ribonuclease T1, which is assumed to interact with Nl of the guanine residue in RNA by a hydrogen bond from the result of X-ray analysis, was changed to alanine by site-directed mutagenesis and its function examined. The nucleolytic activity of the Ala 46 mutant enzyme against pGpC decreased to 0.4% of that of the wild-type enzyme, on the other hand its activity against pApC increased. This result suggests that the Glu 46 is essential for the recognition of the guanine base but that it also interferes with the recognition of the adenine base.

Alanine↗
PubMed1988

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering.

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.

Amino Acid Sequence↗
PubMed1987

Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A.

Ribonuclease T1 (RNase T1, EC 3.1.27.3) is a guanosine-specific ribonuclease that cleaves the 3',5'-phosphodiester linkage of single-stranded RNA. It is assumed that the reaction is generated by concerted acid-base catalysis between residues Glu-58 and His-92 or His-40. From the results of chemical modification and NMR studies, it appeared that the residue Glu-58 was indispensable for nucleolytic activity. However, we have recently demonstrated that Glu-58 is an important but not an essential residue for catalytic activity, using the methods of genetic engineering to change Glu-58 to Gln-58 etc [Nishikawa, S., Morioka, H., Fuchimura, K., Tanaka, T., Uesugi, S., Ohtsuka, E., & Ikehara, M. (1986) Biochem. Biophys. Res. Commun. 138, 789-794]. In the present paper, we report that mutants of RNase T1 with residue Ala-40 or Ala-92 have almost no activity, while mutants that contain Ala-58 retain considerable activity. These results show that the two histidine residues, His-40 and His-92, but not Glu-58, are indispensable for the catalytic activity of the enzyme. We propose a revised reaction mechanism in which two histidine residues play a major role, as they do in the case of RNase A.

Aspergillus oryzae↗
PubMed1987

Crystallographic characterization of recombinant human interleukin 2.

Recombinant human interleukin 2 produced by Escherichia coli was purified to homogeneity and crystallized after being separated from methionyl interleukin 2. Crystals suitable for structural studies have been obtained by the seed enlargement technique, using the method of vapor diffusion with ammonium sulfate as the precipitant at pH 4.6. The space group is P2(1)2(1)2 with cell dimensions a = 49.2 A, b = 87.6 A and c = 32.4 A. The asymmetric unit contains one molecule of the protein. From preliminary results, the crystals are moderately stable to X-rays and produce measurable reflections to a resolution of about 2.2 A. The diffraction data for the native crystals have been collected on a diffractometer at 2.4 A resolution.

Crystallography↗
PubMed1984

The molecular structure of a DNA-triostin A complex.

The molecular structure of triostin A, a cyclic octadepsipeptide antibiotic, has been solved complexed to a DNA double helical fragment with the sequence CGTACG (C, cytosine; G, guanine; T, thymine; A, adenine). The two planar quinoxaline rings of triostin A bis intercalate on the minor groove of the DNA double helix surrounding the CG base pairs at either end. The alanine residues form hydrogen bonds to the guanines. Base stacking in the DNA is perturbed, and the major binding interaction involves a large number of van der Waals contacts between the peptides and the nucleic acid. The adenine residues in the center are in the syn conformation and are paired to thymine through Hoogsteen base pairing.

Crystallization↗
PubMed1984

AT base pairs are less stable than GC base pairs in Z-DNA: the crystal structure of d(m5CGTAm5CG).

Two hexanucleoside pentaphosphates , 5-methyl and 5-bromo cytosine derivatives of d( CpGpTpApCpG ) have been synthesized, crystallized, and their three-dimensional structure solved. They both form left-handed Z-DNA and the methylated derivative has been refined to 1.2 A resolution. These are the first crystal Z-DNA structures that contain AT base pairs. The overall form of the molecule is very similar to that of the unmethylated or the fully methylated (dC-dG)3 hexamer although there are slight changes in base stacking. However, significant differences are found in the hydration of the helical groove. When GC base pairs are present, the helical groove is systematically filled with two water molecules per base pair hydrogen bonded to the bases. Both of these water molecules are not seen in the electron density map in the segments of the helix containing AT base pairs, probably because of solvent disorder. This could be one of the features that makes AT base pairs form Z-DNA less readily than GC base pairs.

Adenine↗
PubMed1983

Molecular structure of d(m5CGTAm5CG): TA pairs in Z-DNA and the consequences.

The hexamer d(m5CGTAm5CG) crystallizes as a left-handed Z-DNA molecule in a isomorphous lattice to d(m5CGm5CGm5CG). The three-dimensional structure has been determined by x-ray diffraction and refined to a final R value of 16% at 1.2A resolution. The molecular structure is similar to the Z-DNA structure seen in d(m5CGm5CGm5CG) and d(CGCGCG). The presence of TpA sequence in the middle of the molecule has resulted in slight changes in the helical twist angles, base pair stacking and twist angles of the base pair planes. The hydration structure is significantly changed from both of methylated and non-methylated d(CGCGCG). A large hole which has no water molecule tightly bound to the DNA molecule is found inside of the deep groove of the DNA helix at the TpA sequence. This deterioration of the hydration structure is associated with destabilization of the Z-DNA structure. These characteristic features of the structure are compared with another Z-DNA hexamer contains TpA sequence, d(br5CGTAbr5CG).

Base Sequence↗
PubMed1982

X-ray diffraction studies of poly(I) . poly(C) analogues with substitution of the 2'-hydroxyl group by a fluorine atom.

From X-ray results of 2'-deoxy-2'-fluoronucleosides, characterization of the sugar conformation was summerized in comparison with the related ribo- and deoxyribonucleosides. It is emphasized that twisted forms of the sugar involving C(4')-exo puckering may be one of the characteristic feature of 2'-deoxy-2'-fluoronucleosides. In context of the characterization, molecular structure of duplex of poly(2'-deoxy-2'-fluoroinosinic acid) and poly(C), poly(dIf1) . poly(C), was analyzed by x-ray fiber diffraction methods. It is shown that the duplex has a strong preference of A'-RNA form than poly (I) . poly (C) and the preference could be interpreted by the sugar conformation of C(3') -endo-C(4')-exo pucker. Comparative studies of X-ray fiber diffraction by poly(I) . poly(C), poly(dIf1) . poly(C), poly(I) . poly(dCf1) and poly (dIf1) . poly (dCf1) are also given.

Deoxyribonucleosides↗
PubMed1981

The crystal and molecular structure of 2'-deoxy-2'-fluoroinosine monohydrate.

The structure of the hydrate of 2'-deoxy-2'-fluoroinosine has been determined by single-crystal x-ray diffraction. The nucleoside crystallizes in space group P2(1)2(1)2(1) with unit cell dimensions, a = 33.291, b = 10. 871, c = 6.897A. There are two nucleosides and two water molecules in the asymmetric unit. The structure was solved by direct methods and refined to a residual R = 0.095. The two independent nucleosides in the asymmetric unit show different conformations about the glycosidic bond, while other structural details are similar. The base orientation to the sugar is syn in molecule A, whereas anti in molecule B. The exocyclic C(4')-C(5') bond conformation defined with respect to C(3')-C(4')-C(5')-O(5') is gauche+ in both molecules A and B. The sugar ring pucker defined by the pseudorotation phase angle P is a twisted conformation in both, C(3')-endo-C(4')-exo with P = 29 degrees in molecule A and C(4')-exo-C(3')-endo with P = 41 degrees in molecule B. It is shown by comparison with x-ray results of other 2'-fluoronucleosides and unmodified nucleosides including inosines that, in addition to a strong preference of the C(3')-endo type pucker, twisted conformations involving C(4')-exo puckering may be one of characteristic features of 2'-fluoronucleosides.

Chemical Phenomena↗