title: Interactions of the 18.5-kDa isoform of myelin basic protein with Ca(2+)-calmodulin: in vitro studies using fluorescence microscopy and spectroscopy.
abstract: The interactions of the 18.5-kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using fluorescence microscopy and spectroscopy. Two forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a hexahistidine-tagged recombinant murine product (rmMBP), with only minor differences in behaviour being observed. Fragments of each protein generated by digestion with cathepsin D (EC 3.4.23.5) were also evaluated. Using fluorescence microscopy, it was shown that MBP and CaM interacted in the presence of Ca2+ under a variety of conditions, including high urea and salt concentrations, indicating that the interaction was specific and not merely electrostatic in nature. Using cathepsin D digestion fragments of MBP, it was further shown that the carboxyl-terminal domain of MBP interacted with Ca(2+)-CaM, consistent with our theoretical prediction. Spectroscopy of the intrinsic fluorescence of the sole Trp residue of MBP showed that binding was cooperative in nature. The dissociation constants for formation of a 1:1 MBP-Ca(2+)-CaM complex were determined to be 2.1 +/- 0.1 and 2.0 +/- 0.2 microM for bMBP/C1 and rmMBP, respectively. Fluorescence spectroscopy using cathepsin D digestion fragments indicated also that the carboxyl-terminal region of each protein interacted with Ca(2+)-CaM, with dissociation constants of 1.8 +/- 0.2 and 2.8 +/- 0.9 microM for the bMBP/C1 and rmMBP fragments, respectively. These values show a roughly 1000-fold lower affinity of MBP for CaM than other CaM-binding peptides, such as myristoylated alanine-rich C-kinase substrate, that are involved in signal transduction.
authors.affiliation: Department of Molecular Biology and Genetics, University of Guelph, ON, Canada.
authors.forename: David S
authors.initials: DS
authors.lastname: Libich
authors.forename: George
authors.initials: G
authors.lastname: Harauz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism; D002417:Cattle; D066298:In Vitro Techniques; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000302:isolation & purification / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D002147:Calmodulin; D004676:Myelin Basic Protein; D020033:Protein Isoforms; D002402:Cathepsin D