title: Improving the stability of the EC1 domain of E-cadherin by thiol alkylation of the cysteine residue.
abstract: The objective of this work was to improve chemical and physical stability of the EC1 protein derived from the extracellular domain of E-cadherin. In solution, the EC1 protein has been shown to form a covalent dimer via a disulfide bond formation followed by physical aggregation and precipitation. To improve solution stability of the EC1 protein, the thiol group of the Cys13 residue in EC1 was alkylated with iodoacetate, iodoacetamide, and maleimide-PEG-5000 to produce thioether derivatives called EC1-IA, EC1-IN, and EC1-PEG. The physical and chemical stabilities of the EC1 derivatives and the parent EC1 were evaluated at various pHs (3.0, 7.0, and 9.0) and temperatures (0, 3, 70 °C). The structural characteristics of each molecule were analyzed by circular dichroism (CD) and fluorescence spectroscopy and the derivatives have similar secondary structure as the parent EC1 protein at pH 7.0. Both EC1-IN and EC1-PEG derivatives showed better chemical and physical stability profiles than did the parent EC1 at pH 7.0. EC1-PEG had the best stability profile compared to EC1-IN and EC1 in solution under various conditions.
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave., Lawrence, KS 66047, USA.
authors.forename: Maulik
authors.initials: M
authors.lastname: Trivedi
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Laurence
authors.forename: Todd D
authors.initials: TD
authors.lastname: Williams
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.forename: Teruna J
authors.initials: TJ
authors.lastname: Siahaan
mesh_terms: D000478:Alkylation; D015820:Cadherins / Q000737:chemistry*; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D007460:Iodoacetamide / Q000737:chemistry; D007461:Iodoacetates / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013440:Sulfides / Q000737:chemistry*; D013696:Temperature
chemical_list: D015820:Cadherins; D007461:Iodoacetates; D008301:Maleimides; D013440:Sulfides; D011092:Polyethylene Glycols; D003545:Cysteine; D007460:Iodoacetamide