Biomedical subjects

T Chatake

Publications and source records attributed to T Chatake.

13 recordsLinked to original sources

PubMed2006

Recent results on hydrogen and hydration in biology studied by neutron macromolecular crystallography.

Neutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins, a technique complimentary to ultra-high-resolution [1, 2] X-ray diffraction. Three different types of neutron diffractometers for biological macromolecules have been constructed in Japan, France and the United States, and they have been used to determine the crystal structures of proteins up to resolution limits of 1.5-2.5 A. Results relating to hydrogen positions and hydration patterns in proteins have been obtained from these studies. Examples include the geometrical details of hydrogen bonds, H/D exchange in proteins and oligonucleotides, the role of hydrogen atoms in enzymatic activity and thermostability, and the dynamical behavior of hydration structures, all of which have been extracted from these structural results and reviewed. Other techniques, such as the growth of large single crystals, the preparation of fully deuterated proteins, the use of cryogenic techniques, and a data base of hydrogen and hydration in proteins, will be described.

Biology↗

PubMed2003

Crystallization of a large single crystal of cubic insulin for neutron protein crystallography.

The growth of a large single crystal of cubic porcine insulin for characterization of hydrogen and hydration in cubic insulin crystals by neutron diffraction analysis is reported. Growth in D2O was investigated based on the phase diagram for cubic insulin to determine appropriate growth conditions, and a large single crystal was then successfully grown by a dialysis method to a size of 4.0 x 4.0 x 1.3 mm3. Neutron diffraction analysis of the cubic insulin crystals was carried out using a single-crystal diffractometer at the JRR-3M reactor of the Japan Atomic Energy Research Institute. In preliminary analysis, Npi appears to be protonated and Ntau deprotonated in His5 in the B-chain, whereas both Npi and Ntau are protonated in His10.

Crystallization↗

PubMed2001

Crystallographic studies on damaged DNAs IV. N( 4)-methoxycytosine shows a second face for Watson-Crick base-pairing, leading to purine transition mutagenesis.

To investigate the mutation mechanism of purine transitions in DNA damaged with methoxyamine, a DNA dodecamer with the sequence d(CGCAAATTmo(4)CGCG), where mo(4)C is 2'-deoxy-N(4)-methoxycytidine, has been synthesized and the crystal structure determined by X-ray analysis. The duplex structure is similar to that of the original undamaged B-form dodecamer, indicating that the methoxylation does not affect the overall DNA conformation. Electron density maps clearly show that the two mo(4)C residues form Watson-Crick-type base pairs with the adenine residues of the opposite strand and that the methoxy groups of mo(4)C adopt the anti conformation to N(3) around the C(4)-N(4) bond. For the pair formation through hydrogen bonds the mo(4)C residues are in the imino tautomeric state. Together with previous work, the present work establishes that the methoxylated cytosine residue can present two alternate faces for Watson-Crick base-pairing, thanks to the amino<-->imino tautomerism allowed by methoxylation. Based on this property, two gene transition routes are proposed.

Adenine↗

PubMed2001

X-ray analyses of d(GCGAAAGC) and d(GXGAAAGCT), where X = 2'-deoxy-5-iodocytidine.

DNA fragments containing a sequence d(GCGAAAGC) are known to be highly thermostable. To investigate the structural basis for such a specific property, crystallographic studies of the DNA octamer and a nanomer d(GXGAAAGCT) (X = 2'-deoxy-5-iodocytidine) have been performed. The present higher resolution X-ray analyses have shown that both DNA oligomers are stabilized respectively to form a zipper-type duplex homodimer. PAGE of these oligomers, however, indicates that they are monomeric even when their crystals were dissolved at room temperature.

Base Sequence↗

PubMed2001

Crystallographic studies on damaged DNAs: III. N(4)-methoxycytosine can form both Watson-Crick type and wobbled base pairs in a B-form duplex.

To investigate the mutation mechanism of purine transition in DNA damaged with methoxyamine, a DNA dodecamer with the sequence d(CGCGAATTmo(4)CGCG), where mo(4)C is 2'-deoxy-N(4)-methoxycytidine, has been synthesized and its crystal structure determined. Two dodecamers form a B-form duplex. Electron density maps clearly show that one of the two mo(4)C residues forms a pair with a guanine residue of the opposite strand, the geometry being the canonical Watson-Crick type, and that the other mo(4)C residue forms a wobble pair with the opposite guanine residue. These two pairings are ascribed to the tautomerization of the methoxylated cytosine moieties between the amino and imino forms.

Base Pairing↗

PubMed2000

X-ray analyses of two DNA dodecamers containing N4-methoxy-cytosine paired with adenine or guanine.

To investigate mismatch of base-pairings in relation to mutagenesis by oxyamines, crystal structures of two DNA dodecamers with the sequence d(CGCZAA TTmo4CGCG) (Z = A or G), containing N4-methoxy-cytosine (mo4C), have been determined by X-ray analysis. These dodecamers essentially form right-handed B-form duplexes, respectively. In the dodecamer with Z = A, the two mo4C residues are adapted in imino form with the anti methoxyl group to form pairs with A on the opposite strand in a manner of Watson-Crick fashion. While in the dodecamer with Z = G, one mo4C in amino form with the anti methoxyl group forms a normal Watson-Crick pair with G, but the other one in imino form with syn methoxyl conformation wobbles with G. Based on these results, possible mutation mechanism has been proposed.

Adenine↗

PubMed1999

Crystallographic studies on damaged DNAs. I. An N(6)-methoxyadenine residue forms a Watson-Crick pair with a cytosine residue in a B-DNA duplex.

Oxyamines such as hydroxylamine and methoxylamine disturb DNA replication and act as potent mutagens, causing nucleotide transition from one purine to another or one pyrimidine to another. In order to investigate mismatch base-pairing in DNA damaged with oxyamines, a dodecamer with the sequence d(CGCGmo(6)AATCCGCG), where mo(6) A is 2'-deoxy-N(6)-methoxyadenosine, was synthesized and its crystal structure determined. No significant conformational changes are found between the present dodecamer and the original undamaged B-form dodecamer. Electron density maps clearly show that the mo(6)A residue forms a base-pair with a 2'-deoxycytidine residue through hydrogen bonds similar to a Watson-Crick G.C base-pair. For these hydrogen bonds to be made, N(6)-methoxyadenine must chemically take the imino form. The methoxylation thus enables the adenine base to mimic a guanine base. As a result, misincorporation of 2'-deoxycytidine instead of thymidine, or 2'-deoxyadenosine instead of 2'-deoxyguanosine, can occur in DNA replication.

Adenine↗

PubMed1999

Crystallographic studies on damaged DNAs. II. N(6)-methoxyadenine can present two alternate faces for Watson-Crick base-pairing, leading to pyrimidine transition mutagenesis.

In a previous paper, 2'-deoxy-N(6)-methoxyadenosine (mo(6)A) was shown to form a mismatch base-pair with 2'-deoxycytidine with a Watson-Crick-type geometry. To fully understand the structural basis of genetic mutations with damaged DNA, it is necessary to examine whether the methoxylated adenine residue still has the ability to form the regular Watson-Crick pairing with a thymine residue. Therefore, a DNA dodecamer with the sequence d(CGCGmo(6)AATTCGCG) has been synthesized and its crystal structure determined. The methoxylation has no significant effect on the overall DNA conformation, which is that of a standard B-form duplex. The methoxylated adenine moieties adopt the amino tautomer with an anti conformation around the C(6)-N(6) bond to the N(1) atom, and they form a Watson-Crick base-pair with thymine residues on the opposite strand, similar to an unmodified adenine residue. It is concluded that methoxylated adenine can present two alternate faces for base-pairing, thanks to the amino<-->imino tautomerism allowed by methoxylation. Based on this property, two gene transition routes are proposed.

Adenine↗

PubMed1999

N6-methoxyadenine in damaged DNA has two faces in property of Watson-Crick base pairing.

In order to investigate mutation mechanism with oxyamine, two DNA dodecamers containing 2'-deoxy-N6-methoxyadenosine have been synthesized and their crystal structures have been determined. The dodecamers are associated in B form duplexes. The methoxy groups attached to the adenine bases do not affect the DNA conformation significantly. Electron densities clearly show that N6-methoxyadenine moiety forms Watson-Crick type pairing with both, thymine and cytosine bases. Such two faces in pairing are the origin of pyrimidine transition mutation.

Adenine↗

PubMed1999

X-ray analysis of a DNA dodecamer containing 2'-deoxy-N4-methoxycytidine.

The crystal structure of DNA dodecamer with the sequence of d(CGCAAATTXGCG), where X is 2'-deoxy-N4-methoxycytidine, has been determined by X-ray analysis. The dodecamers form a double helix with B-form conformation. The electron density indicates that the two modified cytosine bases respectively make a pair with the adenine bases on the opposite strand in a manner of Watson-Crick geometry and that the methoxy groups are in anti conformation to the N3 atom.

Base Pairing↗

PubMed1999

Crystallization and preliminary analysis of a DNA dodecamer of d(CGCGmo6AATCCGCG) containing 2'-deoxy-N6-methoxyadenosine: change in crystal packing with different humidity.

The DNA dodecamer of (CGCGmo6AATCCGCG) containing 2'-deoxy-N6-methoxyadenosine has been crystallized for X-ray analysis in order to investigate the effects of the modified adenosine on base pairing. It has been found that the crystal changes from one form to another during data collection in a manner similar to a phase transition. The two crystal structures show that this phenomenon, ascribed to differences in humidity, is correlated with a change in the contact angle between the two duplexes.

Crystallization↗

PubMed1997

X-ray analysis of DNA dodecamer containing 2'-deoxy-N6-methoxyadenosine.

Oxyamines have been known as a mutagen which attacks amino groups of DNA bases. It is expected that the modified adenine derivative has a tautomer which can form a stable base pair not only with thymine but also with cytosine. For establishing such tautomerization mutagenesis, we have solved a crystal structure of DNA dodecamer containing 2'-deoxy-N6-methoxyadenosine. It is shown that the N6-methoxyadenine takes a imino form to form a Watson-Crick type pairing with cytosine in the DNA duplex.

Base Composition↗

PubMed1995

Structural composition of hammerhead ribozymes.

Eleven kinds of hammerhead ribozymes were designed and synthesized to investigate the structural composition of the complexes and to find suitable crystallization conditions, the substrate chains having been modified to prevent hydrolysis. Electrophoresis patterns indicated that the two strands except for the substrate chain form a binary complex and that they form a ternary complex when mixed with the substrate chain. Both complexes were crystallized. The crystal of the binary complex belongs to a Laue symmetry of 32 (space group of P321, P3(1)21, or P3(2)21) with cell dimensions of a = b = 53.4 and c = 59.4 A. The volume per one nucleotide allows the asymmetric unit to contain one binary complex. Our results suggest that the catalytic part forms a rigid ribozyme structure which induces a scissile reaction when the substrate is bound, in a similar manner to an enzyme protein.

Base Sequence↗