Reversible folding of Ada protein (O6-methylguanine-DNA methyltransferase) of Escherichia coli

Debasish Bhattacharyya, Tapas Hazra, W. David Behnke, Parkson L G Chong, Alexander Kurosky, James Lee, Sankar Mitra

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Abstract

The multifunctional 39 kDa Escherichia coli Ada protein (O6- methylguanine-DNA methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric globular polypeptide with two distinct alkylacceptor activities located in two domains. The two domains are of nearly equal size and are connected by a hinge region. The Ada protein accepts stoichiometrically the alkyl group from O6-alkylguanine in DNA at the Cys- 321 residue and from alkyl phosphotriester at the Cys-69 residue. This protein functions in DNA repair by direct dealkylation of mutagenic O6- alkylguanine. The protein methylated at Cys-69 becomes a transcriptional activator of the genes in the ada regulon, including its own. Each of the two domains functions independently as an alkyl acceptor. The purified homogeneous protein is unstable at 37 °C and spontaneously loses about 30% of its secondary structure in less than 30 min concomitant with a complete loss of activity. However, sedimentation equilibrium studies indicated that the inactive protein remains in the monomeric form without aggregation. Furthermore, electrospray mass spectroscopic analysis indicated the absence of oxidation of the inactive protein. This temperature-dependent inactivation of the Ada protein is inhibited by DNA. In the presence of increasing concentrations of urea or guanidine, the protein gradually loses more than 80% of its structure. The two alkyl acceptor activities appear to be differentially sensitive to unfolding and the phosphotriester methyltransferase activity is resistant to 7 M urea. The partial or complete unfolding induced by urea or guanidine is completely reversed within seconds by removal of the denaturant. The heat-coagulated protein can also be restored to full activity by cycling it through treatment with 8 M urea or 6 M guanidine. These results suggest that the nascent or unfolded Ada polypeptide folds to a metastable form which is active and that the thermodynamically stable structure is partially unfolded and inactive.

Original languageEnglish (US)
Pages (from-to)1722-1730
Number of pages9
JournalBiochemistry
Volume37
Issue number6
DOIs
StatePublished - Feb 10 1998

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Protein Folding
Methyltransferases
Escherichia coli
DNA
Proteins
Urea
Guanidine
Genes
Dealkylation
O-(6)-methylguanine
Regulon
Peptides
Escherichia coli Proteins
Spectroscopic analysis
DNA Repair
Hinges
Sedimentation
Hot Temperature
Repair
Agglomeration

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bhattacharyya, D., Hazra, T., David Behnke, W., Chong, P. L. G., Kurosky, A., Lee, J., & Mitra, S. (1998). Reversible folding of Ada protein (O6-methylguanine-DNA methyltransferase) of Escherichia coli. Biochemistry, 37(6), 1722-1730. https://doi.org/10.1021/bi971852n

Reversible folding of Ada protein (O6-methylguanine-DNA methyltransferase) of Escherichia coli. / Bhattacharyya, Debasish; Hazra, Tapas; David Behnke, W.; Chong, Parkson L G; Kurosky, Alexander; Lee, James; Mitra, Sankar.

In: Biochemistry, Vol. 37, No. 6, 10.02.1998, p. 1722-1730.

Research output: Contribution to journalArticle

Bhattacharyya, D, Hazra, T, David Behnke, W, Chong, PLG, Kurosky, A, Lee, J & Mitra, S 1998, 'Reversible folding of Ada protein (O6-methylguanine-DNA methyltransferase) of Escherichia coli', Biochemistry, vol. 37, no. 6, pp. 1722-1730. https://doi.org/10.1021/bi971852n
Bhattacharyya D, Hazra T, David Behnke W, Chong PLG, Kurosky A, Lee J et al. Reversible folding of Ada protein (O6-methylguanine-DNA methyltransferase) of Escherichia coli. Biochemistry. 1998 Feb 10;37(6):1722-1730. https://doi.org/10.1021/bi971852n
Bhattacharyya, Debasish ; Hazra, Tapas ; David Behnke, W. ; Chong, Parkson L G ; Kurosky, Alexander ; Lee, James ; Mitra, Sankar. / Reversible folding of Ada protein (O6-methylguanine-DNA methyltransferase) of Escherichia coli. In: Biochemistry. 1998 ; Vol. 37, No. 6. pp. 1722-1730.
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abstract = "The multifunctional 39 kDa Escherichia coli Ada protein (O6- methylguanine-DNA methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric globular polypeptide with two distinct alkylacceptor activities located in two domains. The two domains are of nearly equal size and are connected by a hinge region. The Ada protein accepts stoichiometrically the alkyl group from O6-alkylguanine in DNA at the Cys- 321 residue and from alkyl phosphotriester at the Cys-69 residue. This protein functions in DNA repair by direct dealkylation of mutagenic O6- alkylguanine. The protein methylated at Cys-69 becomes a transcriptional activator of the genes in the ada regulon, including its own. Each of the two domains functions independently as an alkyl acceptor. The purified homogeneous protein is unstable at 37 °C and spontaneously loses about 30{\%} of its secondary structure in less than 30 min concomitant with a complete loss of activity. However, sedimentation equilibrium studies indicated that the inactive protein remains in the monomeric form without aggregation. Furthermore, electrospray mass spectroscopic analysis indicated the absence of oxidation of the inactive protein. This temperature-dependent inactivation of the Ada protein is inhibited by DNA. In the presence of increasing concentrations of urea or guanidine, the protein gradually loses more than 80{\%} of its structure. The two alkyl acceptor activities appear to be differentially sensitive to unfolding and the phosphotriester methyltransferase activity is resistant to 7 M urea. The partial or complete unfolding induced by urea or guanidine is completely reversed within seconds by removal of the denaturant. The heat-coagulated protein can also be restored to full activity by cycling it through treatment with 8 M urea or 6 M guanidine. These results suggest that the nascent or unfolded Ada polypeptide folds to a metastable form which is active and that the thermodynamically stable structure is partially unfolded and inactive.",
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