title: Acid-induced formation of molten globule states in the wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase and its three mutated forms: G96A, A183T and G96A/A183T.
abstract: Recent advances in protein chemistry have led to progress in the understanding of protein folding and properties of possible intermediates during the folding of proteins. The molten globule (MG) state, a major intermediate of protein folding, has a denatured state with native-like secondary structure. In the present work, the acid-induced unfolding of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and its three different variants (G96A, A183T and G96A/A183T) were studied by far- and near-UV circular dichroism (CD), intrinsic fluorescent emission spectroscopy and 1-anilino naphthalene-8-sulfonate (ANS) binding. At pH < 3.0, these EPSPS variants acquire partially folded state, which show the characteristics of the MG state, e.g., a drastic reduction of defined tertiary structure and almost no change in the secondary structure. ANS binding experiments show that hydrophobic surface of these variants is exposed to a greater extent in comparison to the native form, at acidic pH. Wild type, G96A, A183T and G96A/A183T acquire MG states at pH 2.0, 1.5, 3.0 and 3.0, respectively, which show that pH stability of MG state of G96A has increased in comparison to wild type; and pH stability of MG states of two other mutants is lower than that of the wild type. The results suggest that there is a direct relationship between stability of protein and pH stability of its folding intermediates.
authors.affiliation: Department of Biochemistry and Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran.
authors.forename: Karimeh
authors.initials: K
authors.lastname: Haghani
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.forename: Ali Hatef
authors.initials: AH
authors.lastname: Salmanian
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
authors.forename: Salar
authors.initials: S
authors.lastname: Bakhtiyari
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000737:chemistry* / Q000235:genetics; D000143:Acids / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays
chemical_list: D000143:Acids; D000817:Anilino Naphthalenesulfonates; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase