Amino Acids

, Volume 39, Issue 3, pp 739–750

The interaction of zinc with membrane-associated 18.5 kDa myelin basic protein: an attenuated total reflectance-Fourier transform infrared spectroscopic study

  • Graham S. T. Smith
  • Lin Chen
  • Vladimir V. Bamm
  • John R. Dutcher
  • George Harauz
Original Article

DOI: 10.1007/s00726-010-0513-7

Cite this article as:
Smith, G.S.T., Chen, L., Bamm, V.V. et al. Amino Acids (2010) 39: 739. doi:10.1007/s00726-010-0513-7

Abstract

Myelin basic protein (MBP) is an essential structural protein required for tight compaction of the myelin sheath of the central nervous system, and belongs to the family of intrinsically disordered proteins. It contains a high proportion of polar and charged amino acids, and has an adaptive conformation depending on its environment and binding surfaces (membranes) or partners (other proteins or small ligands including divalent cations). Zinc is an important stabilizing component of myelin and its concentration is substantially higher than that of any other trace element in the brain. In this study, we investigate the effect of zinc on different variants of 18.5 kDa MBP, including new recombinant forms lacking hexahistidine tags which would interfere with the binding of the cation. Isothermal titration calorimetry showed the dissociation constant to be in the micromolar range for all variants. Circular dichroism spectroscopy showed that there was minimal effect of zinc on the secondary structure on MBP in aqueous solution. When MBP was reconstituted with myelin-mimetic membranes, attenuated total reflectance-Fourier transform infrared spectroscopy revealed that there was a rearrangement of secondary structure components upon addition of zinc that was subtly different for each variant, indicative of a synergistic protein–membrane–cation interaction.

Keywords

Myelin basic protein (MBP) Intrinsically disordered protein Induced folding Isothermal titration calorimetry (ITC) Circular dichroism (CD) Fourier transform infrared (FTIR) spectroscopy Attenuated total reflectance (ATR) spectroscopy 

Abbreviations

ATR

Attenuated total reflectance

CD

Circular dichroism

CNS

Central nervous system

Cyt-LUV

Large unilamellar vesicle with lipid composition, of inner oligodendrocyte membrane leaflet

ddH2O

Double-distilled water

EDTA

Ethylenediamine tetraacetic acid

FTIR

Fourier transform infrared

HEPES

N-(2-Hydroxyethyl) piperazine-N′-2-ethanesulfonic acid

HPLC

High-performance liquid chromatography

IDP

Intrinsically disordered protein

IEX

Ion-exchange (chromatography)

IPTG

Isopropyl-β-d-thiogalactopyranoside

ITC

Isothermal titration calorimetry

MBP

Myelin basic protein

OD

Optical density

PAGE

Polyacrylamide gel electrophoresis

PCR

Polymerase chain reaction

rmC1

Recombinant murine 18.5 kDa MBP, unmodified (LEH6-tagged)

rmC8

Recombinant murine 18.5 kDa MBP, pseudo-deiminated (LEH6-tagged)

SDS

Sodium dodecyl sulphate

UT-rmC1

Untagged recombinant murine 18.5 kDa MBP, unmodified

UT-rmC8

Untagged recombinant murine 18.5 kDa MBP, pseudo-deiminated

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Graham S. T. Smith
    • 1
  • Lin Chen
    • 1
    • 2
  • Vladimir V. Bamm
    • 1
  • John R. Dutcher
    • 2
  • George Harauz
    • 1
  1. 1.Department of Molecular and Cellular BiologyUniversity of GuelphGuelphCanada
  2. 2.Department of PhysicsUniversity of GuelphGuelphCanada

Personalised recommendations