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Tadashi Ueda

Publications and source records attributed to Tadashi Ueda.

At least 19 recordsLinked to original sources
PubMed2006

Amyloid formation in denatured single-mutant lysozymes where residual structures are modulated.

Reduced hen lysozyme has a residual structure involving long-range interaction. It has been demonstrated that a single mutation (A9G, W62G, W111G, or W123G) in the residual structure differently modulates the long-range interactions of reduced lysozyme. To examine whether such variations in the residual structure affect amyloid formation, reduced and alkylated mutant lysozymes were incubated under the amyloid-fibrillation condition. From the analyses of CD spectra and thioflavine T fluorescences, it was suggested that variation in residual structure led to different amyloid formation. Interestingly, the extent of amyloid formation did not always correlate with the extent to which the residual structure was maintained, resulting in the involvement of a hydrophobic cluster normally contained in W111 in the reduced lysozyme.

Amyloid↗
PubMed2006

The functional role of arginine 901 at the C-terminus of the human anion transporter band 3 protein.

To determine which arginine residues are responsible for band 3-mediated anion transport, we analyzed hydroxyphenylglyoxal (HPG)-modified band 3 protein in native erythrocyte membranes. HPG-modification leads to inhibition of the transport of phosphoenolpyruvate, a substrate for band 3-mediated transport. We analyzed the HPG-modified membranes by reverse phase-HPLC, and determined that arginine 901 was modified by HPG. To determine the role of Arg 901 in the conformational change induced by anion exchange, we analyzed HPG-modification of the membranes when 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethypyrocarbonate (DEPC) was present. DNDS and DEPC fix band 3 in the outward and inward conformations, respectively. HPG-modification was unaffected in the presence of DEPC but decreased in the presence of DNDS. In addition to that, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which specifically reacts with the outward conformation of band 3, did not react with HPG-modified membranes. Furthermore, we expressed a band 3 mutant in which Arg 901 was replaced by alanine (R901A) on yeast membranes. The kinetic parameters indicated that the R901A mutation affected the rate of conformational change of the band 3 protein. From these results, we conclude that the most C-terminal arginine, Arg 901, has a functional role in the conformational change that is necessary for anion transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2006

Analysis of internal motions of RNase T1 complexed with a productive substrate involving 15N NMR relaxation measurements.

The backbone dynamics of RNase T1 in the presence of exo-guanosine 2',3'-cyclophosphorothioate (exo-cGPS isomer), which is a productive substrate, and in the presence of 3'-guanylic acid (3'GMP), which is an nonproductive substrate, were examined using (15)N nuclear magnetic resonance. Although the X-ray crystal structure suggests that the modes of binding of these substrates to the active-site cleft are very similar, the order parameters in a number of regions in RNase T1 complexed with exo-cGPS isomer were different from those with 3'GMP. Moreover, the chemical exchange in line width observed for RNase T1 complexed with exo-cGPS isomer was also different from that observed for RNase T1 complexed with 3'GMP. From these results, we concluded that the internal motions in RNase T1 complexed with a productive substrate were not always identical to those in RNase T1 complexed with a nonproductive substrate.

Cyclic GMP↗
PubMed2005

Effect of W62G mutation of hen lysozyme on the folding in vivo.

In previous paper, we showed that W62G mutation caused ill effects at the early stages of folding of the reduced hen lysozyme in vitro. Here, we investigated whether the single mutation brings about drastic turn to in vivo folding of lysozyme. W62G lysozyme was secreted from yeast cells and then purified with ion-exchange chromatography. From the results of gel chromatography and peptide analysis, the species with two cysteines, Cys80 and Cys94, and non-native cystine, Cys64-Cys76, was partially present in secreted product of yeast containing gene for W62G lysozyme. Thus, it was suggested that W62G mutation also affected the in vivo folding of lysozyme.

Amino Acid Substitution↗
PubMed2005

Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site.

PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed.

Amino Acid Sequence↗
PubMed2005

Effect of the structure of the denatured state of lysozyme on the aggregation reaction at the early stages of folding from the reduced form.

We previously demonstrated that the hydrophobic clusters present in hen lysozyme under denaturing conditions were disrupted by the mutation of Trp62 to Gly (W62G). In order to examine the effects of the structure of the denatured state of W62G lysozyme on folding, we analyzed the early events in the folding of reduced W62G lysozyme in detail. From the exchange measurements of disulfide bonds using the variants containing a pair of cysteine residues (1SS), it was found that the formation of disulfide bond in the W62G1SS lysozyme was not accompanied by a prominent interaction between amino acid residues, indicating that the disruption of the hydrophobic core led to the random folding at the early stages in the process of folding of the reduced lysozyme. On the other hand, analyses of the oxidative-renaturation of reduced W62G lysozymes, as well as measurements of the extent of aggregation of the reduced and carboxy amido methylated W62G lysozyme, indicated that the formation of an aggregate is more prominent in the reduced W62G lysozyme than in the reduced wild-type lysozyme. Moreover, a lag phase was detected in the oxidative-renaturation of reduced W62G lysozyme, as based on observations of the recovery of activity. The simulation of the folding process indicated that intermediates were present at the early stages in the folding of the reduced W62G lysozyme. These results suggest that the presence of the intermediates was derived from the random folding at the early stages in the folding process of reduced W62G lysozyme due to the disruption of the structure of the denatured state. Folding thus appears to have been kinetically delayed by these processes, which then led to the significant aggregation of reduced lysozyme. Moreover, from the analysis of amyloid aggregation of the reduced lysozymes, it was suggested that the disruption of the residual structure in denatured state by W62G mutation deterred the formation of the amyloid fibrils of lysozyme.

Amyloid↗
PubMed2005

Amyloid-beta fibril formation is not necessarily required for microglial activation by the peptides.

There is increasing evidence that microglial activation has pathogenic influence on Alzheimer's disease. According to in vitro studies, microglia activated by amyloid-beta (Abeta) peptides have been reported to damage or kill neurons by the release of neurotoxic molecules such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, nitric oxide or reactive oxygen species. Although the relationship between the aggregational state of Abeta peptides and their neurotoxic activities has been well investigated, little is known about the relationship between the aggregational state of Abeta peptides and their ability to induce microglial activation. In the present study, we thus performed both structural and biochemical studies to clarify the relationship between the aggregational state of Abeta peptides and their ability to activate microglia. Our results have shown that, in the presence of interferon-gamma, the Abeta25-35(M(35)Nle) peptide had almost the same potency of activating microglia and producing TNF-alpha as the Abeta25-35 peptide on both protein and mRNA levels, in spite of the fact that former peptide represented much less amyloid fibril formation than the latter in a thioflavine-T fluorometric assay. These results suggest that Abeta fibril formation is not necessarily required for microglial activation by the peptides.

Alzheimer Disease↗
PubMed2005

Characterization of refolded hen lysozyme variant lacking two outside disulfide bonds.

We characterized a refolded hen lysozyme variant containing only two SS-bonds, C64-C80 and C76-C94 (4CAHEL). From CD spectra and its activity, it was found that the refolded 4CAHEL has a structural topology analogous to wild-type lysozyme (WTHEL). Moreover, the refolded 4CAHEL showed no thermal transition, indicating that it had a character like a molten globule.

Animals↗
PubMed2004

Analysis of the early stage of the folding process of reduced lysozyme using all lysozyme variants containing a pair of cysteines.

Twenty-eight hen lysozyme variants that contained a pair of cysteines were constructed to examine the formation of the individual native and nonnative disulfide bonds. We analyzed the extent of the formation of a disulfide bond in each lysozyme variant using a redox buffer (pH 8) containing 1.0 mM reduced and 0.1 mM oxidized glutathione in the absence or presence of 6 M guanidine hydrochloride. In the presence of 6 M guanidine hydrochloride, the extent of the formation of the disulfide bond in each lysozyme variant was proportional to the distance between cysteine residues, indicating that reduced hen lysozyme under a highly denaturing condition adopted a randomly coiled structure. In aqueous solution, the formations of all disulfide bonds occurred much more easily than under a denatured condition. This finding indicated that reduced lysozyme had a somewhat compact structure. Moreover, the scattering data for the extents of the formation of the disulfide bonds among all lysozyme variants were observed. These results suggested that the nonrandom folding occurred in the early stage of the folding of reduced lysozyme, which should provide new insight into the early-stage events in the folding process of reduced lysozyme.

Animals↗
PubMed2004

Determination of the complete cDNA sequence, construction of expression systems, and elucidation of fibrinolytic activity for Tapes japonica lysozyme.

The lysozyme of the marine bivalve, Tapes japonica (13.8 kDa), belongs to the invertebrate lysozyme family and displays both chitinase and isopeptidase activities. We determined the complete cDNA sequence and constructed effective expression systems for this enzyme using Escherichia coli (BL21) and Pichia pastoris. The native and recombinant proteins indicated lysozyme activity and isopeptidase activity, including the proteolysis of d-dimer, a plasminolytic product of stabilized polymeric fibrin. These results will be utilized for the structural and functional study of invertebrate lysozymes, and for the development of applications for thrombosis therapies.

Amino Acid Sequence↗
PubMed2004

[Analysis of the interaction between DnaA protein which is involved in the initiation of chromosomal replication in E. coli and its specifically binding DNA].

DnaA protein binds specifically to a group of binding sites called DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. DnaA domain IV comprises 94 amino acid residues and is required for DNA binding. At first, the backbone assignments of DnaA domain IV in the complexes were determined using several nuclear magnetic resonance (NMR) spectra. Using NMR analysis, we investigated the interaction between DnaA domain IV and DnaA box R1. The 1H-15N HSQC spectrum of DnaA domain IV showed prominent chemical shift perturbations on six residues (Arg399, Ala404, Leu422, Asp433, Thr435 and Thr436). Through homology modeling, we located all of these residues on one side of the surface of the DnaA domain IV molecule. Moreover, we confirmed that these residues in DnaA domain IV bind to DnaA box R1 by mutation analysis. Finally, we compared the chemical shift perturbation of the 1H-15N HSQC spectrum in the presence of the DnaA box with that in the presence of a non-specific oligonucleotide that has a reduced affinity for DnaA, and the results suggested that Leu422 imparts specificity in binding with DnaA box R1.

Bacterial Proteins↗
PubMed2003

Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5'-GMP: structural basis for alterations in substrate specificity.

Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5'-GMP, were determined at resolutions higher than 2 A. In the N71T-5'-GMP and N71S-5'-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.

Crystallography, X-Ray↗
PubMed2003

A method for the detection of asparagine deamidation and aspartate isomerization of proteins by MALDI/TOF-mass spectrometry using endoproteinase Asp-N.

A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-alpha-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of (15)N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.

Amides↗
PubMed2002

Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain.

DnaA protein binds specifically to a group of binding sites collectively called as DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. The DNA-binding domain of DnaA, domain IV, comprises the C-terminal 94 amino acid residues of the protein. We overproduced and purified a protein containing only this domain plus a methionine residue. This protein was stable as a monomer and maintained DnaA box-specific binding activity. We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments. We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six alpha-helices and no beta-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the alpha-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins.

Amino Acid Sequence↗
PubMed2002

A metal binding in the polypeptide chain improves the folding efficiency of a denatured and reduced protein.

In order to examine the effect of a metal binding to the polypeptide chain on the aggregation of a protein in the refolding process, we prepared a mutant hen lysozyme possessing the same Ca(2+) binding site as in human alpha-lactalbumin by Escherichia coli expression system (Ser(-1) CaB lysozyme). In the presence of 2 mM CaCl(2), the refolding yield of Ser(-1) CaB lysozyme at a low protein concentration (25 microg/mL) was similar to that of the wild-type lysozyme (80%), but that at high protein concentration (200 microg/mL) decreased (15%) due to aggregation comparing to that of the wild-type lysozyme (45%). However, the refolding yield of Ser(-1) CaB lysozyme in the presence of 100 mM CaCl(2) even at a protein concentration of 200 microg/mL was 80% and was higher than that of the wild-type lysozyme. From analysis of chemical shift changes of the cross peaks in the backbone region of total correlated spectroscopy (TOCSY) spectra of a decapeptide possessing the same calcium binding site as in Ser(-1) CaB lysozyme in the presence of various concentrations of Ca(2+), it was suggested that the dissociation constant of Ca(2+)-peptide complex was estimated to be 20-36 mM. Moreover, the solubility of the denatured Ser(-1) CaB lysozyme in the presence of 100 mM CaCl(2) was higher than that in the presence of 2 mM CaCl(2) whereas the solubility of the denatured Ser(-1) lysozyme in the presence of 100 mM CaCl(2) was not higher than that in the presence of 2 mM CaCl(2). Therefore, it was concluded that the reduced lysozyme possessing the Ca(2+) binding site was efficiently folded in the presence of high concentration of Ca(2+) (100 mM) even at high protein concentration due to depression of aggregation by the binding of Ca(2+) to the polypeptide chain in Ser(-1) CaB lysozyme.

Animals↗
PubMed2002

Long-range interactions within a nonnative protein.

Protein folding and unfolding are coupled to a range of biological phenomena, from the regulation of cellular activity to the onset of neurodegenerative diseases. Defining the nature of the conformations sampled in nonnative proteins is crucial for understanding the origins of such phenomena. We have used a combination of nuclear magnetic resonance (NMR) spectroscopy and site-directed mutagenesis to study unfolded states of the protein lysozyme. Extensive clusters of hydrophobic structure exist within the wild-type protein even under strongly denaturing conditions. These clusters involve distinct regions of the sequence but are all disrupted by a single point mutation that replaced residue Trp62 with Gly located at the interface of the two major structural domains in the native state. Thus, nativelike structure in the denatured protein is stabilized by the involvement of Trp62 in nonnative and long-range interactions.

Amino Acid Substitution↗
PubMed2002

A nucleotide switch in the Escherichia coli DnaA protein initiates chromosomal replication: evidnece from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo.

The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp- and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.

Adenosine Triphosphate↗