Backbone dynamics of the 18.5 kDa isoform of myelin basic protein reveals transient alpha-helices and a calmodulin-binding site
David S. Libich 1 and George Harauz 1*
1 University of Guelph
* To whom correspondence should be addressed. E-mail: gharauz{at}uoguelph.ca.
Submitted on November 16, 2007
Revised on January 17, 2008
Accepted on 8 February 2008
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Abstract |
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The 18.5 kDa isoform of myelin basic protein (MBP) is the predominant form in adult human central nervous system myelin. It is an intrinsically disordered protein that functions both in membrane adhesion, and as a linker connecting the oligodendrocyte membrane to the underlying cytoskeleton; its specific interactions with calmodulin and SH3-domain containing proteins suggest further multifunctionality in signalling. Here, we have used multidimensional heteronuclear NMR spectroscopy to study the conformational dependence on environment of the protein in aqueous solution (100 mM KCl) and in a membrane-mimetic solvent (30% TFE-d2), particularly to analyse its secondary structure using chemical shift indexing (CSI), and to investigate its backbone dynamics using 15N spin relaxation measurements. Collectively, the data revealed three major segments of the protein with a propensity towards alpha-helicity that was stabilised by membrane-mimetic conditions: T33-D46, V83-T92, and T142-L154 (murine 18.5 kDa sequence numbering). All of these regions corresponded with bioinformatics predictions of ordered secondary structure. The V83-T92 region comprises a primary immunodominant epitope that had previously been shown by site-directed spin labelling and electron paramagnetic resonance spectroscopy to be alpha-helical in membrane-reconstituted systems. The T142-L154 segment overlapped with a predicted calmodulin-binding site. Chemical shift perturbation experiments using labelled MBP and unlabelled calmodulin demonstrated a dramatic conformational change in MBP upon association of the two proteins, and were consistent with the C-terminal segment of MBP being the primary binding site for calmodulin.
Key Words:
calmodulin-binding protein, chemical shift indexing, intrinsically disordered protein, myelin basic protein, relaxation, solution NMR spectroscopy
