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Yuji Kobayashi

Publications and source records attributed to Yuji Kobayashi.

48 records · Page 3Linked to original sources
PubMed2002

The [Lys(-2)-Arg(-1)-des(17-21)]-endothelin-1 peptide retains the specific Arg(-1)-Asp8 salt bridge but reveals discrepancies between NMR data and molecular dynamics simulations.

The [des(17-21)]-endothelin-1 (CSH-ET) and [Lys(-)(2)-Arg(-)(1)-des(17-21)]-endothelin-1 (KR-CSH-ET) peptides, designed by removing the five-residue hydrophobic tail from the endothelin-1 (ET-1) and [Lys(-)(2)-Arg(-)(1)]-endothelin-1 (KR-ET-1) peptides, respectively, were synthesized. Previous studies on KR-ET-1 showed that, in contrast to ET-1, this engineered compound displays a pH-dependent conformational change related to the formation of a stabilizing salt bridge between the Arg(-)(1) and Asp(8) side chains. CD and NMR spectra indicate that CSH-ET and KR-CSH-ET display conformational behavior similar to those of ET-1 and KR-ET-1, respectively. The short salt bridge-stabilized KR-CSH-ET peptide therefore appears to be an attractive elementary scaffold for drug design. The solution structure of the salt-bridged form of KR-CSH-ET was determined by NMR at pH 4.5 and is very similar to the corresponding form of the parent KR-ET-1 peptide. Molecular dynamics simulations of the salt-bridged form of KR-CSH-ET were performed using both the GB/SA implicit solvation scheme or an explicit solvation and the particle-mesh Ewald method for long-range electrostatic calculation. Unexpectedly, the Arg(-)(1)-Asp(8) salt bridge does not display in the simulation the stability that could be expected from the experimental data. The cooperative involvement of a cation-pi interaction in formation of the salt bridge has been hypothesized. Difficulties in accurately simulating cation-pi interactions might be responsible for the lack of stability in the simulation. At this time, however, no definitive explanation for the observed discrepancy between experiments and simulations is available, and further experimental studies appear to be necessary to fully understand in atomic detail the pH-dependent conformational change observed in the KR-ET-1 series.

Amino Acid Motifs↗
PubMed2002

Solution structure of ascidian trypsin inhibitor determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of ascidian trypsin inhibitor (ATI), a 55 amino acid residue protein with four disulfide bridges, was determined by means of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy. The resulting structure of ATI was characterized by an alpha-helical conformation in residues 35-42 and a three-stranded antiparallel beta-sheet in residues 22-26, 29-32, and 48-50. The presence of an alpha-helical conformation was predicted from the consensus sequences of the cystine-stabilized alpha-helical (CSH) motif, which is characterized by an alpha-helix structure in the Cys-X(1)-X(2)-X(3)-Cys portion (corresponding to residues 37-41), linking to the Cys-X-Cys portion (corresponding to residues 12-14) folded in an extended structure. The secondary structure and the overall folding of the main chain of ATI were very similar to those of the Kazal-type inhibitors, such as Japanese quail ovomucoid third domain (OMJPQ3) and leech-derived tryptase inhibitor form C (LDTI-C), although ATI does not show extensive sequence homology to these inhibitors except for a few amino acid residues and six of eight half-cystines. On the basis of these findings, we realign the amino acid sequences of representative Kazal-type inhibitors including ATI and discuss the unique structure of ATI with four disulfide bridges.

Amino Acid Sequence↗
PubMed2002

Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA.

Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form from silkworm ribosomes and tested for their interactions with each other and with RNA fragments corresponding to the GTPase-associated domain of residues 1030-1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro. Mixing of P1 and P2 formed the P1-P2 heterodimer, as demonstrated by gel mobility shift and chemical crosslinking. This heterodimer, but neither P1 or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1-P2)2, assumed from its molecular weight derived from sedimentation analysis. Complex formation strongly stimulated binding of P0 to the GTPase-associated RNA domain. The protein complex and eL12 (E.coli L11-type), which cross-bound to the E.coli equivalent RNA domain, were tested for their function by replacing with the E.coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S subunits. Both P1 and P2, together with P0 and eL12, were required to activate ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation factors as well as eEF-2-dependent GTPase activity. The results suggest that formation of the P1-P2 heterodimer is required for subsequent formation of the P0(P1-P2)2 complex and its functional rRNA binding in silkworm ribosomes.

Amino Acid Sequence↗
PubMed2002

Solution structure of intracellular signal-transducing peptide derived from human beta(2)-adrenergic receptor.

The structure of intracellular third loop peptide (betaIII-2: RRSSKFCLKEKKALK) was studied by CD and NMR spectroscopy. According to the CD study, this peptide forms a helix in the TFE solution. The three-dimensional molecular structure in TFE was determined by the 2D (1)H NMR spectroscopy. The structure consists of a positive charge cluster on the C-terminal side of the peptide chain. This part will be an active site of the peptide interacting with the G-protein.

Amino Acid Sequence↗
PubMed2002

Topography of evoked potentials associated with illusory motion perception as a motion aftereffect.

Motion aftereffect (MAE) is a type of motion illusion. After visual focusing on an object moving in one direction, an illusory perception of motion in the opposite direction occurs while the object suddenly stops moving. In this study we explored components and distribution of evoked potentials related to this motion illusion using MAE caused by motion of concentric rings. When a single array of moving rings was placed to straddle right and left visual fields, a significant bilateral increase of a positive component at about 160 ms (P160) was observed in the occipitotemporal region at the time subjects perceived the motion illusion; this increase was most prominent in the right posterior temporal region. Thus, an early positive component P160 occurs in relation to motion illusion, in agreement with previous results concerning perception of actual motion. When stimuli were presented to produce MAE limited to either the right or left visual hemifield, we also observed a P160 distributed mainly in the right temporal and parietal region. A significant increase in this component was observed in the right posterior temporal region with left hemifield stimulation, while no significant increase was observed with right hemifield stimulation. The right hemispheric dominance of P160 seemed to result partly from functional specialization of the right hemisphere, but hemispheric differences in attentional mechanisms also might contribute to the asymmetric distribution of P160.

Adult↗
PubMed2002

Cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability.

Cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best-characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution. Experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation. Heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive investigation of the biological process by which the functional structure is formed. Mutagenesis and structure analyses based on comparative studies using a thermophile Hydrogenobacter thermophilus cytochrome c-552 and its mesophilic counterpart have provided substantial clues to the mechanism underlying protein stability at the amino-acid level. The molecular mechanisms underlying protein maturation, folding, and stability in bacterial cytochromes c are beginning to be understood.

Amino Acid Sequence↗
PubMed2002

Thermodynamic characterization of variants of mesophilic cytochrome c and its thermophilic counterpart.

Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Delta(DeltaG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.

Bacteria↗
PubMed2002

Novel trimeric adenylate kinase from an extremely thermoacidophilic archaeon, Sulfolobus solfataricus: molecular cloning, nucleotide sequencing, expression in Escherichia coli, and characterization of the recombinant enzyme.

A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.

Adenylate Kinase↗
PubMed2001

Crystallization and preliminary X-ray crystallographic studies of a mutant of ribosome recycling factor from Escherichia coli, Arg132Gly.

Ribosome recycling factor (RRF) plays a central role during the recycling of ribosomes in the final step of protein biosynthesis in prokaryotes and is therefore a favourable target for the development of new antibiotics. The crystal structure of Escherichia coli RRF has been reported to have an open L-shaped conformation, while other RRFs from thermophilic bacteria have a strict L-shaped conformation [Yun et al. (2000), Acta Cryst. D56, 84-85]. Wild-type E. coli RRF has so far not been crystallized free from bound detergent. Here, a mutant of RRF, Arg132Gly, has been crystallized without any detergent. A complete data set from a crystal of this mutant obtained by the hanging-drop vapour-diffusion method has been collected at 2.2 A resolution using synchrotron radiation at 100 K. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 46.02, b = 49.27, c = 49.37 A, beta = 110.1 degrees. The currently refined structure indicates that RRF has a tRNA-like L-shaped conformation.

Crystallization↗