The Single-Ring Thermoanaerobacter brockii Chaperonin 60 (Tbr-EL7) Dimerizes to Tbr-EL14.cntdot.Tbr-ES7 under Protein Folding Conditions Academic Article uri icon

abstract

  • Chaperone proteins assist in the folding of some newly synthesized proteins and inhibit protein aggregation. The Thermoanaerobacter brockii chaperonin proteins (Tbr-EL and Tbr-ES) have recently been purified and characterized [Truscott, W.N., Høj, P. B., & Scopes, R. K. (1994) Eur. J. Biochem. 222, 277-284]; Tbr-EL was a single seven-membered toroid, unlike most GroELs which exist as double toroids. Using high-resolution gel filtration chromatography, we have resolved the purified Tbr-EL into single ringed (Tbr-EL7) and double ringed (Tbr-EL14) species. The latter contained tightly bound Tbr-ES co-chaperonin (Tbr-EL14.Tbr-ES7). In the presence of Mg.ATP and either Escherichia coli GroES (Eco-ES) or Tbr-ES (i.e., under protein folding conditions), the isolated Tbr-EL7 rapidly dimerized to the Tbr-EL14.Eco-ES7 or Tbr-EL14.Tbr-ES7 complexes. The doubly toroidal species thus formed contained > or = 6 molecules tightly bound ADP and one GroES7 and are similar to the asymmetric chaperonin complex isolated from Thermus thermophilus [Taguch, H., Konishi, J., Ishii, N., & Yoshida, M. (1991) J. Biol. Chem. 266, 22411-22418]. The isolated Tbr-EL7 and Tbr-EL14.Tbr-ES7 hydrolyzed ATP at approximate to 2 and 1 min-1, respectively. Addition of a molar excess of Eco-ES7 to the isolated Tbr-EL7 reduced the ATPase activity to 1 min-1, consistent with the formation of Tbr-EL14.Eco-ES7. Eco-ES7 failed to inhibit the Tbr-El14.Tbr-ES7 complex. The isolated Tbr-EL14.Tbr-ES7 complex did not support the folding of Rubisco under nonpermissive conditions. Only when the complex was supplemental with additional GroES was folding of Rubisco observed; i.e., one molar equivalent of GroES was not sufficient for folding. Both Tbr-EL7 and Tbr-EL14.Tbr-ES7 bound on unfolded [35S] Rhodospirillum rubrum Rubisco per mole particle. In contrast, Eco-EL14 bound 2 mol of protein per mole particle, consistent with each toroid having a peptide binding site. Eco-EL14.Eco-ES7 complex only bound one unfolded protein, thus GroES binding blocks one GroEL peptide binding site. Addition of Eco-ES7 to a Eco-EL14.Rubisco2 complex did not result in the displacement of one molecule of Rubisco but in the formation of a ternary Eco-EL14.Rubisco2.Eco-ES7 complex.

authors

  • Todd, Matthew J
  • Walke, Stefan
  • Lorimer, George
  • Truscott, Kaye
  • Scopes, Robert K

publication date

  • November 1995

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