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Construction and characterization of the hetero-oligomer of the group II chaperonin from the hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Extremophiles volume 13, pages 437–445 (2009)Cite this article

Abstract

The hyperthermophilic archaeon Thermococcus sp. strain KS-1 (T. KS-1) expresses two different chaperonin subunits, α and β, for the folding of its proteins. The composition of the subunits in the hexadecameric double ring changes with temperature. The content of the β subunit significantly increases according to the increase in temperature. The homo-oligomer of the β subunit, Cpnβ, is more thermostable than that of the α subunit, Cpnα. Since Cpnα and Cpnβ also have different protein folding activities and interactions with prefoldin, the hetero-oligomer is thought to exhibit different characteristics according to the content of subunits. The hetero-oligomer of the T. KS-1 chaperonin has not been studied, however, because the α and β subunits form hetero-oligomers of varying compositions when they are expressed simultaneously. In this study, we characterized the T. KS-1 chaperonin hetero-oligomer, Cpnαβ, containing both α and β in the alternate order, which was constructed by the expression of α and β subunits in a coordinated fashion and protease digestion. Cpnαβ protected citrate synthase from thermal aggregation, promoted the folding of acid-denatured GFP in an ATP-dependent manner, and exhibited an ATP-dependent conformational change. The yield of refolded GFP generated by Cpnαβ was almost equivalent to that generated by Cpnβ but lower than that generated by Cpnα. In contrast, Cpnαβ exhibited almost the same level of thermal stability as Cpnα, which was lower than that of Cpnβ. The affinity of Cpnαβ to prefoldin was found to be between those of Cpnα and Cpnβ, as expected.

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Abbreviations

T. KS-1:

Hyperthermophilic archaeon Thermococcus sp. strain KS-1

CCT:

Chaperonin containing t-complex-polypeptide 1

PhPFD:

Pyrococcus horikoshii prefoldin

CS:

Citrate synthase from porcine heart

GFP:

Green fluorescence protein

CD:

Circular dichroism spectroscopy

SPR:

Surface plasmon resonance

PAGE:

Polyacrylamide gel electrophoresis

Cpnα:

T. KS-1 chaperonin α homo-oligomer

Cpnβ:

T. KS-1 chaperonin β homo-oligomer

Cpnαβ:

T. KS-1 chaperonin hetero-oligomer containing α and β, alternately

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Acknowledgments

The authors thank Dr. Ryo Iizuka (University of Tokyo) for technical advice. The work reported here is a part of the support program for improving the graduate school education of the “Human Resource Development Program for Scientific Powerhouse,” which is financially supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan, through Tokyo University of Agriculture & Technology. This work was also supported by grants-in-aids for scientific research (17028013, 19370038 and 20059013) and a grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Sports, and Culture of Japan to MY. TK is a recipient of a research fellowship from the Japan Society for the Promotion of Scientist for Young Scientists (19-7771).

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  1. Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan

    Muhamad Sahlan, Taro Kanzaki & Masafumi Yohda

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  1. Muhamad Sahlan
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  2. Taro Kanzaki
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  3. Masafumi Yohda
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Correspondence to Masafumi Yohda.

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Communicated by L. Huang.

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Sahlan, M., Kanzaki, T. & Yohda, M. Construction and characterization of the hetero-oligomer of the group II chaperonin from the hyperthermophilic archaeon, Thermococcus sp. strain KS-1. Extremophiles 13, 437–445 (2009). https://doi.org/10.1007/s00792-009-0229-3

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  • DOI: https://doi.org/10.1007/s00792-009-0229-3

Keywords

  • Thermococcus chaperonin
  • Hetero-oligomer
  • Thermal stability
  • Interaction with prefoldin