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Structures of thermolabile mutants of human glutathione transferase P1-1

Journal Article


Abstract


  • An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix α6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the α-helical propensity of the isolated α6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37°C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue. (C) 2000 Academic Press.

Publication Date


  • 2000

Citation


  • Rossjohn, J., McKinstry, W. J., Oakley, A. J., Parker, M. W., Stenberg, G., Mannervik, B., . . . Aceto, A. (2000). Structures of thermolabile mutants of human glutathione transferase P1-1. Journal of Molecular Biology, 302(2), 295-302. doi:10.1006/jmbi.2000.4054

Scopus Eid


  • 2-s2.0-0034665733

Start Page


  • 295

End Page


  • 302

Volume


  • 302

Issue


  • 2

Abstract


  • An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix α6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the α-helical propensity of the isolated α6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37°C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue. (C) 2000 Academic Press.

Publication Date


  • 2000

Citation


  • Rossjohn, J., McKinstry, W. J., Oakley, A. J., Parker, M. W., Stenberg, G., Mannervik, B., . . . Aceto, A. (2000). Structures of thermolabile mutants of human glutathione transferase P1-1. Journal of Molecular Biology, 302(2), 295-302. doi:10.1006/jmbi.2000.4054

Scopus Eid


  • 2-s2.0-0034665733

Start Page


  • 295

End Page


  • 302

Volume


  • 302

Issue


  • 2