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Masatsune Kainosho

Publications and source records attributed to Masatsune Kainosho.

10 recordsLinked to original sources
PubMed2003

Distinctive solution conformation of phosphatase inhibitor CPI-17 substituted with aspartate at the phosphorylation-site threonine residue.

We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.

Amino Acid Substitution↗
PubMed2003

Rotational diffusion tensor of nucleic acids from 13C NMR relaxation.

Rotational diffusion properties have been derived for the DNA dodecamer d(CGCGAATTCGCG)(2) from (13)C R(1rho) and R(1) measurements on the C(1'), C(3'), and C(4') carbons in samples uniformly enriched in (13)C. The narrow range of C-H bond vector orientations relative to the DNA axis make the analysis particularly sensitive to small structural deviations. As a result, the R(1rho)/R(1) ratios are found to fit poorly to the crystal structures of this dodecamer, but well to a recent solution NMR structure, determined in liquid crystalline media, even though globally the structures are quite similar. A fit of the R(1rho)/R(1) ratios to the solution structure is optimal for an axially symmetric rotational diffusion model, with a diffusion anisotropy, D(\|)/D(perpendicular), of 2.1+/-0.4, and an overall rotational correlation time, (2D(\|)+4D(perpendicular))(-1), of 3.35 ns at 35 degrees C in D(2)O, in excellent agreement with values obtained from hydrodynamic modeling.

Carbon Isotopes↗
PubMed2003

The NMR studies of substituent effects on the N-H...N hydrogen bond in duplex DNA using 2'-deoxynebularine and 15N labeled 5-substituted-2'-deoxyuridine base pairs.

The effects of substitutions on various NMR parameters, which may influence the hydrogen bond strengths of Watson-Crick base pairs, were investigated for DNA dodecamers containing 5-substituted-2'-deoxyuridine derivatives and 2'-deoxynebularine in the oligomers, 5'-d(CGCGNA TX CGCG)-3'; where N and X are 2'-deoxy nebularine and [N3-(15)N]-2'-deoxyuridine derivatives. The substitution effects on NMR parameters were linearly correlated with the pKa values of the 2'-dexoyuridine derivatives.

Base Pairing↗
PubMed2002

Identification of the metal ion binding site on an RNA motif from hammerhead ribozymes using (15)N NMR spectroscopy.

An RNA oligomer, r(GGACGAGUCC), which mimics the metal ion-binding motif of hammerhead ribozymes, was shown to fold by itself into a conformation possessing a metal ion binding property which is similar to that of the intact ribozyme (Tanaka, et al. J. Am. Chem. Soc. 2000, 122, 11303-11310). To determine the metal ion-binding site of this motif at an atomic level, we synthesized a series of RNA oligomers which were selectively labeled with a (15)N-labeled guanosine at each of the four guanosine residues. The (15)N-chemical shift perturbation with Cd(II) ions by one-dimensional (1D) (15)N NMR spectra showed that the chemical shift of the N7 of the G7 residue, N7/G7, in the metal ion-binding motif was specifically perturbed. This is the first experimental evidence to prove that the N7/G7 binds with a Cd(II) ion.

Binding Sites↗
PubMed2002

Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca(2+)-ATPase: probing nucleotide binding by multidimensional NMR.

The skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1a) mediates muscle relaxation by pumping Ca(2+) from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a.

Adenosine Triphosphate↗
PubMed2002

Graphical analysis of the relative orientation of molecular alignment tensors for a protein dissolved in two different anisotropic media.

In order to determine precise three-dimensional structures of proteins by residual dipolar coupling constants as the major or even exclusive structural constraints, it is essential to use two anisotropic media. In doing so, a reliable and versatile method for estimating the relative orientation of the alignment tensors for the molecules dissolved in different anisotropic media is required. In this communication, we present a new graphical approach for this purpose, which does not require structural information of the target molecules. The correlation map for the two independent data sets of residual dipolar coupling constants, which can be obtained for the molecules in different anisotropic media, strongly depends on the relative orientation of the alignment tensors. We have simulated the correlation maps for all possible combinations of the Euler angles, which transform one alignment tensor to the other, and compared them to the experimental data sets reported for labeled human ubiquitin. This simple graphical method affords a useful starting point for the structural determinations using residual dipolar couplings.

Anisotropy↗