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

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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.

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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

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Acknowledgments

This work was supported by the Canadian Institutes of Health Research (MOP #74468, to G.H. and Vladimir Ladizhansky), the Natural Sciences and Engineering Research Council of Canada (G.H.), and the Advanced Foods and Materials Network Centre of Excellence (G.H. and J.R.D.). G.S.T.S. and V.V.B. are the recipients of a Doctoral Studentship and a Postdoctoral Fellowship, respectively, from the Multiple Sclerosis Society of Canada. G.S.T.S. was initially the recipient of a stipend from an anonymous private donor in support of this work. J.R.D. acknowledges support from the Canada Research Chair Program. The authors are grateful to Drs. Mumdooh Ahmed, Vladimir Ladizhansky, and Leonid Brown (Physics, University of Guelph) for their advice on ATR-FTIR data collection and analysis, and to Dr. Joan Boggs (Hospital for Sick Children, Toronto) and Dr. Jeremy Lee (University of Saskatoon) for comments on the manuscript.

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada

    Graham S. T. Smith, Lin Chen, Vladimir V. Bamm & George Harauz

  2. Department of Physics, University of Guelph, Guelph, ON, N1G 2W1, Canada

    Lin Chen & John R. Dutcher

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  1. Graham S. T. Smith
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  2. Lin Chen
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  3. Vladimir V. Bamm
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  5. George Harauz
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Correspondence to George Harauz.

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G. S. T. Smith, L. Chen and V. V. Bamm contributed equally to this work.

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Smith, G.S.T., Chen, L., Bamm, V.V. et al. The interaction of zinc with membrane-associated 18.5 kDa myelin basic protein: an attenuated total reflectance-Fourier transform infrared spectroscopic study. Amino Acids 39, 739–750 (2010). https://doi.org/10.1007/s00726-010-0513-7

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