Biomedical subjects

T Imoto

Publications and source records attributed to T Imoto.

At least 73 records · Page 4Linked to original sources

PubMed1997

Analysis of the stabilization of hen lysozyme by helix macrodipole and charged side chain interaction.

In the N-terminal region of the alpha-helix of the c-type lysozymes, two Asx residues exist at the 18th and 27th positions. Hen lysozyme has Asp18/Asn27 (18D/27N), and we prepared three mutant lysozymes, Asn18/Asn27 (18N/27N), Asn18/Asp27 (18N/27D), and Asp18/Asp27 (18D/27D). The stability of the wild-type (18D/27N) lysozyme supported the existence of a hydrogen bond between the side chain of Asp18 and the amide group at the N1 position in the alpha-helix, while the stability of the 18N/27D lysozyme supported the presence of the capping box between the Ser24 (N-cap) and Asp27 residues. Although electrostatic repulsion was observed between Asp18 and Asp27 residues in 18D/27D lysozyme, the dissociation of each residue contributed to stabilizing the B-helix in 18D/27D lysozyme through hydrogen bonding and charge-helix macrodipole interaction. This is the first evidence that two neighboring negative charges at the N-terminus of the helix both increased the stability of the protein.

Amino Acid Sequence↗

PubMed1997

Influence of mutations of the N-cap residue, Gly4, on stability and structure of hen lysozyme.

Hen lysozyme, with three alpha-helices (A, B, and C), is a c-type lysozyme. In these lysozymes, Ser24 and Asp88 located at the N-cap position in the B- and C-helix, respectively, are mostly conserved, but residue 4 at the N-cap position in A-helix is variable. To investigate the effect of mutation at position 4 on the stability of hen lysozyme, we prepared five mutant lysozymes and examined their stabilities and structures. Gly4Pro lysozyme (G4P), in which Gly4 was replaced by Pro, was less stable by 8.8 kJ/mol than the wild-type lysozyme, possibly because the side chain at position 7 is shifted away from the A-helix. The other mutant lysozymes were of almost equal stability to the wild-type lysozyme, although the hydrogen bonds of the amide groups at positions N1-N3 in the A-helix were absent or altered. These results indicated that various mutations at the N-cap position in the A-helix would be allowed as long as the negative charge of Glu7 at the N-terminus stabilized the A-helix.

Amino Acid Sequence↗

PubMed1997

Analysis of the stability of mutant lysozymes at position 15 using X-ray crystallography.

His 15 of hen lysozyme is located at the protein surface and is partly buried by the neighboring residues. The side chain of His 15 forms hydrogen bonds with surrounding residues and these hydrogen bonds are somewhat buried. A series of mutant lysozymes at the position 15 (Gly, Ala, Val, and Phe) was prepared, and their stabilities were analyzed by GdnHCl denaturation and X-ray crystallography. The mutants were less stable than the wild type at pH 5.5 and 35 degrees C. In H15G and H15A, X-ray crystallography revealed two fixed water molecules at the mutated region, which formed similar hydrogen bonds to those in the wild type. On the other hand, it was suggested that the hydrogen bonds were disrupted and that several unfavorable van der Waals' contacts occurred in H15V and H15F. Therefore, we concluded that His 15 stabilized the lysozyme structure by forming hydrogen bonds and the best packing with the neighboring residues. Moreover, we found that the method of protein stabilization by increasing the hydrophobicity of an amino acid residue was not always effectively applicable, especially when the residue had formed a hydrogen bond.

Animals↗

PubMed1997

An improved method for preparing lysozyme with chemically 13C-enriched methionine residues using 2-aminothiophenol as a reagent of thiolysis.

Jones et al. have reported that the epsilon-carbons of methionine residues in myoglobin can be enriched with stable isotope (13C) in two steps, i.e., methylation of methionine residues with 13CH3I in the protein and thiolysis using dithiothreitol [Jones, W.C., Rothgeb, T.M., and Gurd, F.R.N. (1976) J. Biol. Chem. 251,7452-7460]. Using their method, we failed to prepare active lysozyme in which the epsilon-carbons of methionine residues are enriched with 13C, because many side reactions took place under the thiolysis condition (pH 10.5, 37 degrees C). When we employed 2-aminothiophenol as a reagent for thiolysis, the reduction proceeded under a weakly acidic condition to afford fully active lysozyme, in which the epsilon-carbons of two methionine residues were enriched with 13C, in a 30% yield. Analysis of the 13C-edited NOESY spectra of 13C-enriched methionine lysozyme in the absence and presence of a substrate analogue indicated the occurrence of conformational change around Met 105 in lysozyme.

Aniline Compounds↗

PubMed1997

Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser.

When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)-lysozyme). The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as the methionine is a hydrophobic amino acid. A Met(-2)Pro(-1) or Met(-2)Ser(-1) sequence was introduced at the N-terminus of hen egg-white lysozyme. The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase, as expected, since the penultimate residue was proline or serine. From the analyses of solubility, stability and refolding yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(-1)-lysozyme) showed closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N-terminus of hen egg-white lysozyme (Pro(-1)-lysozyme). Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy and its activity were almost identical with those of native hen egg-white lysozyme.

Animals↗

PubMed1997

Lack of gurmarin sensitivity of sweet taste receptors innervated by the glossopharyngeal nerve in C57BL mice.

Effects of a sweet response inhibitor, gurmarin, on responses of the chorda tympani and glossopharyngeal nerves were studied in the C57BL/KsJ strain of mice. The lingual application of gurmarin at 3.0 microg/ml (approxiamtely 0.7 microM) or more significantly suppressed chorda tympani responses to 0.5 M sucrose, as previously reported. The magnitude of gurmarin inhibition of the chorda tympani responses reached a plateau (approximately 45% of control) at 50 microg/ml (approximately 11.9 microM). In contrast, no such gurmarin inhibition of sucrose responses was observed in the glossopharyngeal nerve even at 100 microg/ml (approximately 23.8 microM). The lingual application of a proteolytic enzyme, pronase, suppressed sucrose responses to <20% of control in both chorda tympani and glossopharyngeal nerves. These results suggest differential sensitivity to gurmarin by sweet taste receptors innervated by the chorda tympani and the glossopharyngeal nerves. The former apparently possess gurmarin sensitivity, whereas most of the latter may be lacking sensitivity.

Animals↗

PubMed1996

Identification of the peptide region that folds native conformation in the early stage of the renaturation of reduced lysozyme.

We prepared three peptide fragments (fg.59-105, fg.63-105 and fg.64-105) by the BrCN cleavage of mutant lysozymes where Ile58, Trp62 and Trp63 were mutated to Met, respectively. From the analysis of formation of the disulfide bonds among Cys64, Cys76, Cys80 and Cys94 in the renaturation of each peptide fragment from the reduced form, Trp62 and Trp63 were required for the effective formation of two disulfide bonds. Especially, Trp62 was found to be involved in the correct formation of the disulfide bonds.

Amino Acid Sequence↗

PubMed1996

Engineering of lysozyme.

As the most extensively investigated model protein, the protein engineering of lysozyme is described. By utilizing modifications made possible by chemical or gene engineering methods, we can get a better understanding of protein behaviour and we can also improve their properties. The results of the protein engineering of lysozyme are described, which give some ideas for a better understanding of the physiological function of proteins, their stabilization, and how to engineer a novel protein.

Animals↗

PubMed1996

Reduced immunogenicity of monomethoxypolyethylene glycol-modified lysozyme for activation of T cells.

Chemical modification of proteins with monomethoxypolyethylene glycol (mPEG) will reduce the immunogenicity of proteins. In the present study, we evaluated the effect of mPEG modification on the capacity of hen egg-white lysozyme (HEL) to stimulate T cells. Lymph node cells (LNCs) from mice immunized with HEL or with mPEG-HEL conjugate were cultured with these antigens, then we measured the proliferation and IL-2 production. mPEG-modification lowered the T cell activating capacity of HEL, both in vitro and in vivo. Neither toxicity, nor antigen non-specific immunosuppressive capacity was observed with mPEG-HEL and unconjugated mPEG. Suppressor cells were unlikely to be generated in the mPEG-HEL-primed LNCs. We next examined the behavior of mPEG-HEL during antigen processing. The capacity of HEL and mPEG-HEL to be incorporated by live cells was much the same. However, the susceptibility to various proteases, including endosomal/lysosomal enzymes, was significantly decreased by mPEG modification. The increased resistance of mPEG-HEL to proteolytic degradation implied that the conjugate was poorly presented to T cells. This may be an important factor related to the low immunogenicity of mPEG modified proteins.

Animals↗

PubMed1996

A mutation study of catalytic residue Asp 52 in hen egg lysozyme.

We constructed a system for the expression and secretion of mature hen lysozyme by yeast using an intermediate "secretion-signal cassette" vector, pKP1700, containing the yeast invertase signal sequence and an expression vector, pAM82, for secretion and maturation of the enzyme. Using this system, mutants of hen lysozyme were produced and the catalytic mechanism in hen lysozyme was definitely confirmed. The hydrolytic activity of D52A as to substrate (NAG)6 at pH 5.0 was obviously decreased to one-four hundredth of that of the wild type. The acidic limb of the pH-activity profile observed for the wild-type was not observed for D52A, and the pKa of Glu 35 on the alkaline limb was seen for both enzymes. Moreover, no structural change was detected on X-ray analysis of D52A. Therefore, we confirmed that dissociated Asp 52 assists catalysis by producing an electrostatic field and by stabilizing the oxocarbonium ion intermediate in the dissociated form.

Animals↗

PubMed1996

Stabilization of lysozyme by introducing N-glycosylation signal sequence.

We designed mutant lysozymes with N-glycosylation signal sequences (Asn48-Gly49-Thr-50 and Asn87-Ile88-Thr89) by substituting Asp to Asn at positions 48 and 87. When these mutant lysozymes were expressed by using yeast (Saccharomyces cerevisiae) in Burkholder minimum medium, N-glycosylation occurred in both lysozymes. The mutant lysozyme with the oligosaccharide at Asn87 showed a similar character to a reported polymannosyl lysozyme [Nakamura, Takasaki, Kobayashi, and Kato (1993) J. Biol. Chem. 268, 12706-12712; Kato, Takasaki, and Ban (1994) FEBS Lett. 355, 76-80]. As judged from the thermodynamic stabilities of the lysozymes obtained by the guanidine hydrochloride denaturation method, the oligosaccharide-bearing mutant lysozymes were more stable by 0.4-1.6 kcal/mol than the corresponding unglycosylated lysozymes. Therefore, it is suggested that the introduction of an N-glycosylation signal sequence into a protein is an effective means to increase the stability of the protein.

Amino Acid Sequence↗

PubMed1996

Analysis of the transition state in the unfolding of hen lysozyme by introduction of Gly-Pro and Pro-Gly sequences at the same site.

We developed a sensitive method for analyzing the conformation of the transition state in the unfolding of hen lysozyme. The activation free energy changes of mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same sites (Gly47Pro47', Pro47Gly47', Gly101Pro102, Pro101Gly102, Gly117Pro118, Pro117Gly118, Gly121Pro122, and Pro121Gly122 lysozymes) were obtained for the unfolding in aqueous solution at pH 5.5 and 35 degrees C. Since we had shown that the difference of energies of the unfolded state in lysozymes having an introduced Gly-Pro or Pro-Gly sequence at the same site was much smaller than the difference of energies of the folded states [Motoshima, H., Ueda, T., Hashimoto, Y., Tsutsumi, M., and Imoto, T. (1995) J. Biochem. 118, 1138-1144], we could estimate the difference of energies of the folded and the transition states unequivocally. We defined the phi-value as the ratio of the difference in the free energy change in the transition state to that in the free energy change in the folded state between lysozymes with Gly-Pro and Pro-Gly sequences at the same site. The phi-values gave information on how much the mutated sites retained the folded structure in the transition state. These values were 0.45 around position 47, which is located in the beta-sheet structure, 0.12 at position 101-102, which is located in the loop at the upper part of the active site, 0.17 at position 117-118, which is located in the beta-turn and 0.64 at position 121-122, which is located in the 3(10)-helix. Therefore, in the transition state in the unfolding of lysozyme, it was found that the 3(10)-helical region had a similar structure to the intact region, while both the beta-turn and the loop at the upper part of the active site were considerably unfolded. The beta-sheet structure was also moderately disrupted in the transition state.

Animals↗

PubMed1996

Situation of monomethoxypolyethylene glycol covalently attached to lysozyme.

We selectively introduced monomethoxypolyethylene glycol (mPEG) 5000, 2000, and 550 into Asp119 in lysozyme. To examine how the mPEGs were present on the surface of the modified lysozyme, the activities, binding abilities to the Fab fragment of anti-lysozyme monoclonal antibody, net charges and nuclear magnetic resonance (NMR) spectra of mPEG lysozymes were examined. With the increase in molecular weight of mPEG, the activities and binding abilities to the Fab of mPEG lysozyme decreased. However, introduced mPEG5000 did not cause complete inhibition of the activities and binding abilities to the Fab, while the maximum length of mPEG5000 was so great that it largely covered the surface of the lysozyme molecule. Analyses of the net charges and NMR suggested that the introduced mPEG preferentially assumed a folded conformation on the surface rather than spread all over the surface. Based on the structure of mPEG lysozyme, the mechanism of the reduced immunogenicity of mPEG lysozyme was discussed.

Antibodies, Monoclonal↗

PubMed1996

Effective renaturation of denatured and reduced immunoglobulin G in vitro without assistance of chaperone.

IgG is an oligomeric protein that consists of two heavy and two light chains. To form the oligomer, a highly concentrated protein would be required on renaturation. On the other hand, refolding of proteins at high concentration often led to aggregation. Therefore, denatured and reduced oligomeric protein scarcely refolded to the native structure. As was expected, the folding yield of the denatured and reduced IgG was below 5% under the condition employed in rapid dilution. The low folding yield was elucidated to be due to assembly or aggregation. Using a renaturation method previously developed to depress aggregation effectively by means of slow dialysis, the refolding yield of the denatured and reduced IgG at above 1 mg/ml was above 70%. Most of the refolded IgG was identical with the intact material based on analyses by affinity chromatography and SDS-PAGE.

Chromatography, Affinity↗

PubMed1996

Effect of additives on the renaturation of reduced lysozyme in the presence of 4 M urea.

Reduced lysozyme was renatured by sulfhydryl-disulfide interchange reactions at pH 8.0 in the presence of 4 M urea, with or without additives at 40 degrees C. In the absence of additives, the final folding yield of reduced lysozyme was approximately 40%. In the presence of sarcosine, glycerol, ammonium sulfate, N-acetyl glucosamine and glucose, its folding yields increased in all cases. In particular, yields increased up to 90% in the presence of 4 M sarcosine. On the other hand, the melting temperatures of lysozyme with or without additives in 0.02 M citrate buffer (pH 6.0) were evaluated using differential scanning calorimetry. In the absence of additive, the melting temperature of lysozyme was 73.8 degrees C. In the presence of additives, all melting temperatures were higher than that of lysozyme in the absence of additives. Moreover, there was a good correlation on addition of additives between an increase in the folding yield of reduced lysozyme with 4 M urea and an increase in the melting temperature without 4 M urea. Therefore, we conclude that additives, which stabilize native lysozyme, are effective at increasing the folding yield of reduced lysozyme in 4 M urea.

Acetylglucosamine↗

PubMed1996

[Establishment of fundamentals for protein research. Stabilization of protein].

Proteins are usually in an equilibrium between the folded and the unfolded state. Therefore, for the stabilization of proteins against reversible denaturation, the free energy change for the unfolding should be increased by stabilizing the folded state by lowering the energy level of the folded state or by destabilizing the unfolded state by raising the energy level of the unfolded state. On the other hand, various processes can be coupled with the unfolded state of proteins. For example, protease digestion of proteins at physiological temperature may be one of such processes. The process would lead to an irreversible denaturation. For the stabilization of proteins against the irreversible denaturation coupled with the unfolded state, a kinetic stabilization is important, that is, the activation free energy for the unfolding should be increased. Methods for the kinetic stabilization were discussed. Finally, the irreversible chemical deterioration of proteins was considered.

Animals↗

PubMed1995

Kinetically trapped structure in the renaturation of reduced oxindolealanine 62 lysozyme.

The refolded products of reduced native lysozyme and reduced OX62 lysozyme, in which Trp62 is converted to oxindolealanine (OX62) during the renaturation of sulfhydryl-disulfide interchange reactions at pH 8 and 37 degrees C, were investigated. On gel-chromatography eluted with 10% aqueous acetic acid containing 4 M urea, two peaks appeared in the refolded product of reduced OX62 lysozyme while a single peak appeared in the refolded product of reduced native lysozyme. From the analyses of the activity and primary and the tertiary structures of the derivative, the structure of the derivative from reduced native lysozyme was confirmed to be identical to that of the untreated one. On the other hand, the refolded product from reduced OX62 lysozyme had the same primary structure but a different tertiary structure compared to the untreated one. The tertiary structure of the refolded product from the reduced OX62 lysozyme was changed to that of the untreated one by the denaturation-renaturation treatment under nonreduced conditions. However, the refolded species was barely changed to that of the untreated one by incubation under physiological conditions. Therefore, the refolded product from reduced OX62 lysozyme was suggested to be a metastable and kinetically trapped product in the renaturation process of reduced OX62 lysozyme. In addition, an interaction involving the folding process of reduced lysozyme was discussed on the basis of the NMR analyses of the metastable structure.

Amino Acid Sequence↗

PubMed1995

Electrophysiological characterization of the inhibitory effect of a novel peptide gurmarin on the sweet taste response in rats.

The effect of an anti-sweet peptide, gurmarin purified from the leaves of Gymnema sylvestre, was studied electrophysiologically on taste responses of the rat chorda tympani. The action of gurmarin was highly specific to sweet taste so that responses to various sweeteners including sugars, sweet amino acids and an artificial sweetener, saccharin were all suppressed. The most effective pH at which the rat tongue was treated with gurmarin was found to be 4.5, which corresponds to the isoelectric point of the peptide. At this condition about 5 microM of gurmarin was sufficient to reveal maximal effect and this was still significant at 0.5 microM (2 micrograms/ml). Although the suppressed responses required several hours to attain complete recovery, anti-gurmarin serum shortened the recovery time considerably. On the other hand, intravenous injection of gurmarin did not cause any significant effects on taste responses at all. These results suggest that gurmarin acts on the apical side of the taste cell, possibly by binding to the sweet taste receptor protein.

Amino Acids↗