Publications (5)17.79 Total impact
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Article: Biochemical characterization and cooperation with co-chaperones of heat shock protein 90 from Schizosaccharomyces pombe.
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ABSTRACT: The characterization of Hsp90 from the fission yeast Schizosaccharomyces pombe was performed. Hsp90 of S. pombe existed as a dimer and exhibited ATP-dependent conformational changes. It captured unfolded proteins in the ATP-free open conformation and protected them from thermal aggregation. Hsp90 of S. pombe was also able to refold thermally denatured firefly luciferase. The co-chaperones Sti1 and Aha1 bound Hsp90 and modulated its activity. Because the affinity of Sti1 was higher than that of Aha1, the effect of Sti1 appeared to dominate when both co-chaperones existed simultaneously.Journal of Bioscience and Bioengineering 05/2013; · 1.79 Impact Factor -
Article: Nonequivalence Observed for the 16-Meric Structure of a Small Heat Shock Protein, SpHsp16.0, from Schizosaccharomyces pombe.
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ABSTRACT: Small heat shock proteins (sHsps) play a role in preventing the fatal aggregation of denatured proteins in the presence of stresses. The sHsps exist as monodisperse oligomers in their resting state. Because the hydrophobic N-terminal regions of sHsps are possible interaction sites for denatured proteins, the manner of assembly of the oligomer is critical for the activation and inactivation mechanisms. Here, we report the oligomer architecture of SpHsp16.0 from Schizosaccharomyces pombe determined with X-ray crystallography and small angle X-ray scattering. Both results indicate that eight dimers of SpHsp16.0 form an elongated sphere with 422 symmetry. The monomers show nonequivalence in the interaction with neighboring monomers and conformations of the N- and C-terminal regions. Variants for the N-terminal phenylalanine residues indicate that the oligomer formation ability is highly correlated with chaperone activity. Structural and biophysical results are discussed in terms of their possible relevance to the activation mechanism of SpHsp16.0.Structure 12/2012; · 6.35 Impact Factor -
Article: StHsp14.0, a small heat shock protein of Sulfolobus tokodaii strain 7, protects denatured proteins from aggregation in the partially dissociated conformation.
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ABSTRACT: The small heat shock protein (sHsp), categorized into a class of molecular chaperones, binds and stabilizes denatured proteins for the purpose of preventing aggregation. The sHsps undergo transition between different oligomeric states to control their nature. We have been studying the function of sHsp of Sulfolobus tokodaii, StHsp14.0. StHsp14.0 exists as 24meric oligomer, and exhibits oligomer dissociation and molecular chaperone activity over 80°C. We constructed and characterized StHsp14.0 mutants with replacement of the C-terminal IKI to WKW, IKF, FKI and FKF. All mutant complexes dissociated into dimers at 50°C. Among them, StHsp14.0FKF is almost completely dissociated, probably to dimers. All mutants protected citrate synthase (CS) from thermal aggregation at 50°C. But, the activity of StHsp14.0FKF was the lowest. Then, we examined the complexes of StHsp14.0 mutants with denatured CS by SAXS. StHsp14.0WKW protects denatured CS by forming the globular complexes of 24 subunits and a substrate. StHsp14.0FKF also formed similar complex but the number of subunits in the complex is a little smaller. These results suggest that the dimer itself exhibits low chaperone activity, and a partially dissociated oligomer of StHsp14.0 protects a denatured protein from interacting with other molecules by surrounding it.Journal of biochemistry 06/2011; 150(4):403-9. · 1.95 Impact Factor -
Article: Adaptation of a hyperthermophilic group II chaperonin to relatively moderate temperatures.
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ABSTRACT: Group II chaperonins exist in archaea and the eukaryotic cytosol, and mediate protein folding in an ATP-dependent manner. We have been studying the reaction mechanism of group II chaperonins using alpha chaperonin, the recombinant chaperonin alpha subunit homo-oligomer from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1 (T. KS-1). Although the high stability and activity of T. KS-1 alpha chaperonin provided advantages for our study, its high thermophilicity caused the difficulty in using various analytical methods. To resolve this problem, we tried to adapt T. KS-1 alpha chaperonin to moderate temperatures by mutations. The comparison of amino acid sequences between 26 thermophilic and 17 mesophilic chaperonins showed that three amino acid replacements are likely responsible for the difference of their optimal temperatures. We introduced three single mutations and also their double combinations into T. KS-1 alpha chaperonin. Among them, K323R single mutant exhibited the improvements of the folding activity and the ATP-dependent conformational change ability at lower temperatures, such as 50 degrees C and 40 degrees C. Since K323 may secure helix 12 in the closed conformation by interacting with D198, the replacement of Lys to Arg likely induced the higher mobility of the built-in lid, resulting in the higher activity at relatively low temperatures.Protein Engineering Design and Selection 02/2010; 23(5):393-402. · 2.94 Impact Factor -
Article: Sequential action of ATP-dependent subunit conformational change and interaction between helical protrusions in the closure of the built-in lid of group II chaperonins.
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ABSTRACT: ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.Journal of Biological Chemistry 11/2008; 283(50):34773-84. · 4.77 Impact Factor
Top Journals
Institutions
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2008–2013
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Tokyo University of Agriculture and Technology
- Division of Biotechnology and Life Science
Tokyo, Tokyo-to, Japan -
Keio University
- Department of Physics
Tokyo, Tokyo-to, Japan
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