Complex of GroEL chaperone with fluorescein-labeled denatured pepsin: stoichiometry and role of the ligands

  • N.Yu. Marchenko V.N. Karazin Kharkov National University
  • V.V. Marchenkov Institute of Protein Research, RAS
  • N.V. Kotova Institute of Protein Research, RAS
  • P.A. Kaliman V.N. Karazin Kharkov National University
  • G.V. Semisotnov Institute of Protein Research, RAS
Keywords: protein-protein interaction, chaperones, GroEL, GroES, fluorescence anisotropy titration

Abstract

One of the main functions of the molecular chaperone GroEL (tetradecameric protein from E.coli cells) is the binding of polypeptides with nonrigid packed side chains and prevention of their nonspecific aggregation. In the present work the interaction of GroEL with fluorescein-labeled nonnative pepsin (pH 7.5) in the absence and in the presence of GroEL various ligands (Mg2+ ions, ADP, ATP and heptameric co-chaperone GroES) has been studied using size exclusion chromatography and fluorescence anisotropy titration. It was shown that Mg2+ ions play determinant role in the formation of the complex between GroEL and nonnative pepsin at moderate ionic strength (100 mM KCl). In the presence of Mg2+ ions adenyl nucleotides (ADP or ATP) decrease the GroEL affinity to nonnative pepsin in the dependence on nucleotide nature, concentration and presence of co-chaperone GroES. Fluorescence anisotropy titration showed that in the presence of Mg2+ ions one GroEL molecule binds two molecules of denatured pepsin with average dissociation constant Kdiss=3.6 •10-9M.

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Author Biographies

N.Yu. Marchenko, V.N. Karazin Kharkov National University

4 Svoboda sq., Kharkov, Ukraine

V.V. Marchenkov, Institute of Protein Research, RAS

4 Institutskaya st., Pushchino, Russia

N.V. Kotova, Institute of Protein Research, RAS

4 Institutskaya st., Pushchino, Russia

P.A. Kaliman, V.N. Karazin Kharkov National University

4 Svoboda sq., Kharkov, Ukraine

G.V. Semisotnov, Institute of Protein Research, RAS

4 Institutskaya st., Pushchino, Russia

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Published
2005-06-06
Cited
How to Cite
Marchenko, N., Marchenkov, V., Kotova, N., Kaliman, P., & Semisotnov, G. (2005). Complex of GroEL chaperone with fluorescein-labeled denatured pepsin: stoichiometry and role of the ligands. Biophysical Bulletin, 1(15), 53-56. Retrieved from https://periodicals.karazin.ua/biophysvisnyk/article/view/17990
Issue
Vol. 1 No. 15 (2005): Біофізичний вісник
Section
Molecular biophysics
Copyright (c) 2005 Biophysical visnik

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