Interactions of the 18.5-kDa isoform of myelin basic protein with ca2+-calmodulin: In vitro studies using fluorescence microscopy and spectroscopy

David S. Libich, George Harauz

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

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 Ca2+-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-Ca2+-CaM complex were determined to be 2.1 ± 0.1 and 2.0 ± 0.2 μM 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 Ca2+-CaM, with dissociation constants of 1.8 ± 0.2 and 2.8 ± 0.9 μM 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.

Original languageEnglish (US)
Pages (from-to)395-406
Number of pages12
JournalBiochemistry and Cell Biology
Volume80
Issue number4
DOIs
StatePublished - Jan 1 2002
Externally publishedYes

Keywords

  • Ca-calmodulin
  • Cathepsin D
  • Intrinsic Trp fluorescence
  • MARCKS
  • Myelin basic protein

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Interactions of the 18.5-kDa isoform of myelin basic protein with ca<sup>2+</sup>-calmodulin: In vitro studies using fluorescence microscopy and spectroscopy'. Together they form a unique fingerprint.

Cite this