title: Carbodiimide EDC induces cross-links that stabilize RNase A C-dimer against dissociation: EDC adducts can affect protein net charge, conformation, and activity.
abstract: RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. These oligomers show high catalytic activity against double-stranded RNA and striking antitumor actions that are lacking in the monomer. However, the dissociation of these metastable oligomers limits their therapeutic potential. Here, a widely used conjugating agent, 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), has been used to induce the formation of amide bonds between carboxylate and amine groups of different subunits of the RNase A C-dimer. A cross-linked C-dimer which does not dissociate was isolated and was found have augmented enzymatic activity toward double-stranded RNA relative to the unmodified C-dimer. Characterization using chromatography, electrophoresis, mass spectrometry, and NMR spectroscopy revealed that the EDC-treated C-dimer retains its structure and contains one to three novel amide bonds. Moreover, both the EDC-treated C-dimer and EDC-treated RNase A monomer were found to carry an increased number of positive charges (about 6 ± 2 charges per subunit). These additional positive charges are presumably due to adduct formation with EDC, which neutralizes a negatively charged carboxylate group and couples it to a positively charged tertiary amine. The increased net positive charge endowed by EDC adducts likely contributes to the heightened cleavage of double-stranded RNA of the EDC-treated monomer and EDC-treated C-dimer. Further evidence for EDC adduct formation is provided by the reaction of EDC with a dipeptide Ac-Asp-Ala-NH(2) monitored by NMR spectroscopy and mass spectrometry. To determine if EDC adduct formation with proteins is common and how this affects protein net charge, conformation, and activity, four well-characterized proteins, ribonuclease Sa, hen lysozyme, carbonic anhydrase, and hemoglobin, were incubated with EDC and the products were characterized. EDC formed adducts with all these proteins, as judged by mass spectrometry and electrophoresis. Moreover, all suffered conformational changes ranging from slight structural modifications in the case of lysozyme, to denaturation for hemoglobin as measured by NMR spectroscopy and enzyme assays. We conclude that EDC adduct formation with proteins can affect their net charge, conformation, and enzymatic activity.
authors.affiliation: Instituto de Química Física Rocasolano (CSIC), Madrid, Spain.
authors.forename: Jorge P
authors.initials: JP
authors.lastname: López-Alonso
authors.forename: Fernando
authors.initials: F
authors.lastname: Diez-García
authors.forename: Josep
authors.initials: J
authors.lastname: Font
authors.forename: Marc
authors.initials: M
authors.lastname: Ribó
authors.forename: Maria
authors.initials: M
authors.lastname: Vilanova
authors.forename: J Martin
authors.initials: JM
authors.lastname: Scholtz
authors.forename: Carlos
authors.initials: C
authors.lastname: González
authors.forename: Francesca
authors.initials: F
authors.lastname: Vottariello
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gotte
authors.forename: Massimo
authors.initials: M
authors.lastname: Libonati
authors.forename: Douglas V
authors.initials: DV
authors.lastname: Laurents
mesh_terms: D002234:Carbodiimides / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019281:Dimerization; D008958:Models, Molecular; D015394:Molecular Structure; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: C557098:1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D012259:Ribonuclease, Pancreatic