Structural Stability of Oligomeric Chaperonin 10: the Role of Two β-Strands at the N and C Termini in Structural Stabilization

Sakane, Isao; Ikeda, Masanobu; Matsumoto, Chiduru; Higurashi, Takashi; Inoue, Katsuaki; Hongo, Kunihiro; Mizobata, Tomohiro; Kawata, Yasushi

doi:10.1016/j.jmb.2004.09.082

Cited by 16 publications

(25 citation statements)

References 35 publications

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“…The unfolding transition of GroES in Gdn-HCl was reported in detail previously. [17][18][19] As shown in Fig. 3a and b, Hhsp10 and Gp31 displayed almost the same stability as GroES, 17 with C m ≈ 0.7-1 M Gdn-HCl.…”

Section: Structural Characteristics Of Hsp10 Proteinsmentioning

confidence: 57%

“…17,19 Recombinant Hhsp10 was constructed from an expression vector of Rhsp10 (pRSC550-hsp10) by changing Gly to Ser at position 52 using a QuikChange site-directed mutagenesis kit (Stratagene). The plasmid pRSC550-hsp10 was a generous gift of Dr. Hideaki Itoh from Akita University.…”

Section: Methodsmentioning

confidence: 99%

“…Cultivation, expression, and purification of Hhsp10 were performed according to the Rhsp10 expression and purification protocol. 19 Gp31 protein was obtained from cells [E. coli BL21 (DE3)] possessing a plasmid [pET23a(+)/gp31] that contained the structural gene for phage T4 gp31 protein. The gene was isolated from bacteriophage T4 using PCR (the phage samples were a generous gift of Dr. Fumio Arisaka from the Tokyo Institute of Technology) and ligated into the NdeI/HindIII restriction sites of pET23a(+) (Novagen).…”

Section: Methodsmentioning

confidence: 99%

“…This C m has been shown to correspond to the structural unfolding of dissociated Tmahsp10 monomers, as reported previously. 19 Acid unfolding was also studied for GroES and Hhsp10 proteins by measuring CD spectra at various pH levels. Acid unfolding transition curves of GroES and Hhsp10 were obtained as shown in Fig.…”

Section: Structural Characteristics Of Hsp10 Proteinsmentioning

confidence: 99%

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Fibril Formation of hsp10 Homologue Proteins and Determination of Fibril Core Regions: Differences in Fibril Core Regions Dependent on Subtle Differences in Amino Acid Sequence

Yagi¹,

Sato²,

Yoshida³

et al. 2008

Journal of Molecular Biology

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Section: Structural Characteristics Of Hsp10 Proteinsmentioning

confidence: 57%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Structural Characteristics Of Hsp10 Proteinsmentioning

confidence: 99%

See 2 more Smart Citations

Fibril Formation of hsp10 Homologue Proteins and Determination of Fibril Core Regions: Differences in Fibril Core Regions Dependent on Subtle Differences in Amino Acid Sequence

Yagi¹,

Sato²,

Yoshida³

et al. 2008

Journal of Molecular Biology

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“…Folding studies of cpn10 from other species (human, rat, T. maritima, and A. aeolicus) have been performed, and it was confirmed that the folding was reversible. [8][9][10] However, there are discrepancies in the unfolding models among studies using different proteins and denaturants. Guidry 8 and Luke 10 have reported that equilibrium unfolding of human cpn10 protein can be expressed by a two-state model and that a denatured heptameric state may be seen in experiments using urea.…”

Section: Introductionmentioning

confidence: 99%

Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

et al. 2008

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It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single-molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force-extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm-like chain model, the contour lengths calculated from the peaks in the profile suggested that linkedGroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.Keywords: force spectroscopy; mechanical unfolding intermediates; protein subunit stability; covalently linked oligomeric protein; GroES; cpn10Abbreviations: AFM, atomic force microscopy; CH-ESC7, ESC7 that has two-cysteine residues and six-histidine residues extending from the N terminus and C terminus, respectively; ESC7, engineered form of cochaperonin GroES with covalently linked subunits; Gdn-HCl, guanidine hydrochloride; PBS, phosphate saline buffer.

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Thermal activation of the co‐chaperonins GroES and gp31 probed by mass spectrometry

Geels

Calmat

Heck

et al. 2008

Rapid Comm Mass Spectrometry

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Many biological active proteins are assembled in protein complexes. Understanding the (dis)assembly of such complexes is therefore of major interest. Here we use mass spectrometry to monitor the disassembly induced by thermal activation of the heptameric co-chaperonins GroES and gp31. We use native electrospray ionization mass spectrometry (ESI-MS) on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer to monitor the stoichiometry of the chaperonins. A thermally controlled electrospray setup was employed to analyze conformational and stoichiometric changes of the chaperonins at varying temperature. The native ESI-MS data agreed well with data obtained from fluorescence spectroscopy as the measured thermal dissociation temperatures of the complexes were in good agreement. Furthermore, we observed that thermal denaturing of GroES and gp31 proceeds via intermediate steps of all oligomeric forms, with no evidence of a transiently stable unfolded heptamer. We also evaluated the thermal dissociation of the chaperonins in the gas phase using infrared multiphoton dissociation (IRMPD) for thermal activation. Using gas-phase activation the smaller (2-4) oligomers were not detected, only down to the pentamer, whereafter the complex seemed to dissociate completely. These results demonstrate clearly that conformational changes of GroES and gp31 due to heating in solution and in the gas phase are significantly different.

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Structural Stability of Oligomeric Chaperonin 10: the Role of Two β-Strands at the N and C Termini in Structural Stabilization

Cited by 16 publications

References 35 publications

Fibril Formation of hsp10 Homologue Proteins and Determination of Fibril Core Regions: Differences in Fibril Core Regions Dependent on Subtle Differences in Amino Acid Sequence

Fibril Formation of hsp10 Homologue Proteins and Determination of Fibril Core Regions: Differences in Fibril Core Regions Dependent on Subtle Differences in Amino Acid Sequence

Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

Thermal activation of the co‐chaperonins GroES and gp31 probed by mass spectrometry

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