Structural Stability of Covalently Linked GroES Heptamer: Advantages in the Formation of Oligomeric Structure

Sakane, Isao; Hongo, Kunihiro; Motojima, Fumihiro; Murayama, Shigeto; Mizobata, Tomohiro; Kawata, Yasushi

doi:10.1016/j.jmb.2007.01.037

Cited by 15 publications

(31 citation statements)

References 39 publications

(53 reference statements)

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“…14 We found that ESC7 displays two-state cooperative transitions in equilibrium unfolding experiments but shows multiphasic kinetic profiles, which indicated the existence of transient intermediates.…”

Section: Resultsmentioning

confidence: 73%

“…Here, it should be noted that in chemical unfolding experiments, we have determined that the covalent linker between the subunits restricts the mobility of the subunits as a whole but does not change the stability of an individual subunit. 14 The remaining DL c value, derived from peaks 2 to 1, was significantly smaller than the theoretical value of unfolded GroES subunit. Although we do not possess additional confirming data, we observed that the net extension of the polymer seen after peak 1, represented by an L c value of 34 nm, was reasonably close to the theoretical length of seven intact GroES subunits each linked by a triglycine linker (the distance between the N and C terminus of an intact GroES subunit is estimated at 3.3 nm from X-ray crystallographic measurement, 6 and the length of a single triglycine linker is about 1.0 nm; these two values yield a total of 3.3 nm Â 7 þ 1.0 nm Â 6 ¼ 29.1 nm).…”

Section: Resultsmentioning

confidence: 76%

“…CH-ESC7 protein was expressed in E. coli using the same procedure for ESC7. 14 Judging by SDS-PAGE analysis, some truncated proteins that were possibly caused by protease degradation from the intact, fulllength CH-ESC7 were also observed. Although we tried to purify CH-ESC7 using nickel-chelate column chromatography, we could not separate the truncated proteins.…”

Section: Proteinsmentioning

confidence: 98%

“…The expression vector of covalently linked GroES (pESC7) 14 was modified as follows to produce a variant of ESC7 suitable for mechanical manipulation. Small DNA fragments were inserted at the SmaI and NaeI sites of the pESC7 vector, which resulted in the formation of a new vector, pCH-ESC7.…”

Section: Proteinsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 76%

Section: Proteinsmentioning

confidence: 98%

Section: Proteinsmentioning

confidence: 99%

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

et al. 2008

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“…Cofactor Concatamers Suggest Preferred Subunit Arrangements-To eliminate uncertainties concerning the heterogeneity of molecular species in the hetero-oligomeric cofactor complexes, various combinations of physically linked subunits were constructed based on the previously reported strategy to produce active GroES as a concatamer of seven subunits (53). In the concatamers, subunits are connected from the C terminus of one subunit to the N terminus of the next subunit via different three amino acid linkers (GSG, AAG, GSS or ASS) (Fig.…”

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confidence: 99%

Chaperonin Cofactors, Cpn10 and Cpn20, of Green Algae and Plants Function as Hetero-oligomeric Ring Complexes

Tsai

Mueller‐Cajar

Saschenbrecker

et al. 2012

Journal of Biological Chemistry

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Background:The chloroplast chaperonin system is encoded by multiple genes. Results: The chaperonin cofactors of the green alga C. reinhardtii and the plant A. thaliana form hetero-oligomeric ring complexes containing seven ϳ10-kDa modules. Conclusion:The hetero-oligomeric cofactors were able to interact with chaperonin and assist protein folding. Significance: Formation of hetero-oligomers can explain the occurrence of multiple chaperonin cofactor genes in chloroplasts.

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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 Covalently Linked GroES Heptamer: Advantages in the Formation of Oligomeric Structure

Cited by 15 publications

References 39 publications

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

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

Chaperonin Cofactors, Cpn10 and Cpn20, of Green Algae and Plants Function as Hetero-oligomeric Ring Complexes

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

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