title: Interaction of the 18.5-kD isoform of myelin basic protein with Ca2+ -calmodulin: effects of deimination assessed by intrinsic Trp fluorescence spectroscopy, dynamic light scattering, and circular dichroism.
abstract: The effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP-Cit(0)), an engineered protein with six quasi-citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP-qCit(6)), human component C1 (hMBP-Cit(0)), and human component C8 (hMBP-Cit(6)), both obtained from a patient with multiple sclerosis (MS). Both rmMBP-Cit(0) and hMBP-Cit(0) bound CaM in a Ca(2+)-dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM-binding. Inherent Trp fluorescence spectroscopy and a single-site binding model were used to determine the dissociation constants: K(d) = 144 +/- 76 nM for rmMBP-Cit(0), and K(d) = 42 +/- 15 nM for hMBP-Cit(0). For rmMBP-qCit(6) and hMBP-Cit(6), the changes in fluorescence were suggestive of a two-site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady-state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination.
authors.affiliation: Department of Molecular Biology and Genetics, Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.
authors.forename: David S
authors.initials: DS
authors.lastname: Libich
authors.forename: Christopher M D
authors.initials: CM
authors.lastname: Hill
authors.forename: Ian R
authors.initials: IR
authors.lastname: Bates
authors.forename: F Ross
authors.initials: FR
authors.lastname: Hallett
authors.forename: Souzan
authors.initials: S
authors.lastname: Armstrong
authors.forename: Aleksander
authors.initials: A
authors.lastname: Siemiarczuk
authors.forename: George
authors.initials: G
authors.lastname: Harauz
mesh_terms: D000178:Acrylamides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D002413:Cations, Divalent; D002942:Circular Dichroism; D002956:Citrulline / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009103:Multiple Sclerosis / Q000378:metabolism; D004676:Myelin Basic Protein / Q000737:chemistry* / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
chemical_list: D000178:Acrylamides; D002147:Calmodulin; D002413:Cations, Divalent; D004676:Myelin Basic Protein; D020033:Protein Isoforms; D002956:Citrulline; D002118:Calcium