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Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

¹ Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russian Federation² Maturation des ARN et Enzymologie Moléculaire UMR 7567 CNRS-UHP B.P. 239, Faculté des Sciences Université Henry Poincaré Nancy I, 54506, Vandoeuvre-lès-Nancy Cedex, France

(RECEIVED November 3, 2004; FINAL REVISION December 15, 2004; ACCEPTED December 31, 2004)

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K_d of 0.4 and 0.9 µM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.

Keywords: GAPDH; GroEL; oligomeric proteins; refolding; denaturation; immobilization; chemical modification; protein–protein interactions

Abbreviations: DAB, 3,5'-diaminobenzidine tetrahydrochloride • DTNB, 5,5'-dithiobis(2-nitrobenzoate) • DSC, differential scanning calorimetry • DTT, dithiothreitol • GAPDH, glyceraldehyde-3-phosphate dehydrogenase • TBS, Tris-buffered saline

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041211205.

Reprint requests to: Vladimir I. Muronetz, Department of Biochemistry of Animal Cell, Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russian Federation; e-mail: vimuronets{at}belozersky.msu.ru; fax: +(095) 939-31-81.

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