Biomedical subjects

Hideto Shimahara

Publications and source records attributed to Hideto Shimahara.

6 recordsLinked to original sources

PubMed2007

Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase.

The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

Binding Sites↗

PubMed2006

Distinct DNA methylation activity of Dnmt3a and Dnmt3b towards naked and nucleosomal DNA.

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. De novo type DNA methyltransferases Dnmt3a and Dnmt3b are responsible for creating DNA methylation patterns during embryogenesis and in germ cells. Although their in vitro DNA methylation properties are similar, Dnmt3a and Dnmt3b methylate different genomic DNA regions in vivo. In the present study, we have examined the DNA methylation activity of Dnmt3a and Dnmt3b towards nucleosomes reconstituted from recombinant histones and DNAs, and compared it to that of the corresponding naked DNAs. Dnmt3a showed higher DNA methylation activity than Dnmt3b towards naked DNA and the naked part of nucleosomal DNA. On the other hand, Dnmt3a scarcely methylated the DNA within the nucleosome core region, while Dnmt3b significantly did, although the activity was low. We propose that the preferential DNA methylation activity of Dnmt3a towards the naked part of nucleosomal DNA and the significant methylation activity of Dnmt3b towards the nucleosome core region contribute to their distinct methylation of genomic DNA in vivo.

Animals↗

PubMed2005

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin.

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.

Aluminum↗

PubMed2005

Assembly and disassembly of nucleosome core particles containing histone variants by human nucleosome assembly protein I.

Histone variants play important roles in the maintenance and regulation of the chromatin structure. In order to characterize the biochemical properties of the chromatin structure containing histone variants, we investigated the dynamic status of nucleosome core particles (NCPs) that were assembled with recombinant histones. We found that in the presence of nucleosome assembly protein I (NAP-I), a histone chaperone, H2A-Barr body deficient (H2A.Bbd) confers the most flexible nucleosome structure among the mammalian histone H2A variants known thus far. NAP-I mediated the efficient assembly and disassembly of the H2A.Bbd-H2B dimers from NCPs. This reaction was accomplished more efficiently when the NCPs contained H3.3, a histone H3 variant known to be localized in the active chromatin, than when the NCPs contained the canonical H3. These observations indicate that the histone variants H2A.Bbd and H3.3 are involved in the formation and maintenance of the active chromatin structure. We also observed that acidic histone binding proteins, TAF-I/SET and B23.1, demonstrated dimer assembly and disassembly activity, but the efficiency of their activity was considerably lower than that of NAP-I. Thus, both the acidic nature of NAP-I and its other functional structure(s) may be essential to mediate the assembly and disassembly of the dimers in NCPs.

Acids↗

PubMed2004

Molecular-orientation analysis based on alignment-induced TROSY chemical shift changes.

We present a new NMR technique for determining the alignment tensor of a weakly aligned protein using only alignment-induced 15N transverse relaxation optimized spectroscopy (TROSY) chemical shift changes. Alignment-induced TROSY chemical shift changes reflect the combined contributions from two different anisotropic spin interactions including the residual dipolar couplings (RDCs) and the residual chemical shift anisotropy effects (RCSAs). We show here that these two residual anisotropic spin interactions' values, encoded in the TROSY chemical shift changes, can be used to determine a weakly aligned protein's alignment tensor. To prove the significance of this method, we show that our TROSY-based analysis gives the consistent alignment angles with those determined using RDCs for 15N-labeled ubiquitin (8.6 kDa) in an aligned medium, within an uncertainty range estimated by considering experimental and structural noises, being 5 degrees at most. Because our approach requires a pre-determined 15N CSA tensor value, we also estimated the uncertainties associated with the resultant alignment tensor values caused by variation in 15N CSA tensors. In spite of the significant variations in literature-reported 15N CSA tensors, they gave consistent orientation angles within an uncertainty range. These results ensure that our TROSY-based approach is a useful alternative to the RDC-based method to determine the alignment angles especially for large proteins in a weakly aligned state.

Anisotropy↗

PubMed2003

Measurement of 15N chemical shift anisotropy in a protein dissolved in a dilute liquid crystalline medium with the application of magic angle sample spinning.

The chemical shifts of nuclei that have chemical shielding anisotropy, such as the 15N amide in a protein, show significant changes in their chemical shifts when the sample is altered from an isotropic state to an aligned state. Such orientation-dependent chemical shift changes provide information on the magnitudes and orientation of the chemical shielding tensors relative to the molecule's alignment frame. Because of the extremely high sensitivity of the chemical shifts to the sample conditions, the changes in chemical shifts induced by adding aligned bicelles do not arise only from the protein alignment but should also include the accumulated effects of environmental changes including protein-bicelle interactions. With the aim of determining accurate 15N chemical shielding tensor values for solution proteins, here we have used magic angle sample spinning (MAS) to observe discriminately the orientation-dependent changes in the 15N chemical shift. The application of MAS to an aligned bicelle solution removes the torque that aligns the bicelles against the magnetic field. Thus, the application of MAS to a protein in a bicelle solution eliminates only the molecular alignment effect, while keeping all other sample conditions the same. The observed chemical shift differences between experiments with and without MAS therefore provide accurate values of the orientation-dependent 15N chemical shifts. From the values for ubiquitin in a 7.5% (w/v) bicelle medium, we determined the 15N chemical shielding anisotropy (CSA) tensor. For this evaluation, we considered uncertainties in measuring the 1H-15N dipolar couplings and the 15N chemical shifts and also structural noise present in the reference X-ray structure, assuming a random distribution of each NH bond vector in a cone with 5 degrees deviation from the original orientation. Taking into account these types of noise, we determined the average 15N CSA tensor for the residues in ubiquitin as Delta sigma=-162.0+/-4.3 ppm, eta=0.18+/-0.02, and beta=18.6+/-0.5 degrees, assuming a 1H-15N bond length of 1.02 A. These tensor values are consistent with those obtained from solid-state NMR experiments.

Amides↗