Effect of Ca2+-Mg2+ exchange on the flexibility and/or conformation of the small domain in monomeric actin

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Abstract

A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F(DA)), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature dyer the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26°C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26°C that was not detected in the case of Mg-G-actin. In the temperature range between 6°C and 26°C the slope of the temperature profile of f' is the same for Ca- G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin.

Original languageEnglish
Pages (from-to)2474-2481
Number of pages8
JournalBiophysical Journal
Volume74
Issue number5
Publication statusPublished - May 1998

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Actins
Temperature
Fluorescence
Fluorescence Resonance Energy Transfer
Proteins
Fluorescein-5-isothiocyanate

ASJC Scopus subject areas

  • Biophysics

Cite this

Effect of Ca2+-Mg2+ exchange on the flexibility and/or conformation of the small domain in monomeric actin. / Nyitrai, M.; Hild, G.; Lakos, Zs; Somogyi, B.

In: Biophysical Journal, Vol. 74, No. 5, 05.1998, p. 2474-2481.

Research output: Contribution to journalArticle

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abstract = "A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F(DA)), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature dyer the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26°C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26°C that was not detected in the case of Mg-G-actin. In the temperature range between 6°C and 26°C the slope of the temperature profile of f' is the same for Ca- G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin.",
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