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Charge isomers of myelin basic protein: structure and interactions with membranes, nucleotide analogues, and calmodulin.

Wang C, Neugebauer U, Bürck J, Myllykoski M, Baumgärtel P, Popp J, Kursula P - PLoS ONE (2011)

Bottom Line: Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1.Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM.Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oulu, Oulu, Finland.

ABSTRACT
As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids--a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.

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Features of MBP interaction with membranes and micelles.A. CD analysis of MBP conformation bound to lipid vesicles. Dotted line, MBP in phosphate buffer; solid line, MBP bound to DMPG/DMPC vesicles at a protein/lipid ratio of 1/300. MRE, mean residue ellipticity. B. The interaction with DPC micelles, as observed by intrinsic tryptophan fluorescence. Both rmC1 (black) and rmC8 (red) have increased Trp fluorescence in the presence (thick lines) of 0.1% DPC, compared to its absence (thin lines). The identical result was observed at temperatures between +25 and +42°C, and in the DPC concentration range 0.1–2.0%. The shift of the peak to a much lower wavelength while increasing its intensity is a sign that the single Trp residue of MBP is buried inside the DPC micelle, in a hydrophobic environment.
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pone-0019915-g005: Features of MBP interaction with membranes and micelles.A. CD analysis of MBP conformation bound to lipid vesicles. Dotted line, MBP in phosphate buffer; solid line, MBP bound to DMPG/DMPC vesicles at a protein/lipid ratio of 1/300. MRE, mean residue ellipticity. B. The interaction with DPC micelles, as observed by intrinsic tryptophan fluorescence. Both rmC1 (black) and rmC8 (red) have increased Trp fluorescence in the presence (thick lines) of 0.1% DPC, compared to its absence (thin lines). The identical result was observed at temperatures between +25 and +42°C, and in the DPC concentration range 0.1–2.0%. The shift of the peak to a much lower wavelength while increasing its intensity is a sign that the single Trp residue of MBP is buried inside the DPC micelle, in a hydrophobic environment.

Mentions: The effects of several membrane mimics, including DPC and other phosphocholines, LDAO, SDS, and TFE, on the secondary structure of MBP were investigated by SRCD (Figure 4). It was pointed out that DPC micelles can constitute a realistic model of membrane interfaces [45], and thus, we decided to study the effect of different concentrations of DPC on the structure of rmMBP. Such experiments were carried out already previously with rmC1 and conventional CD spectroscopy [46], at much lower protein concentration. With the addition of DPC, rmC1 acquired a significant fraction of helical secondary structure (Figure 4A). In addition, all the three distinctive helical peaks increased with increasing DPC concentration from 25 mM to 100 mM. The effect of DPC on rmC8 is similar to rmC1 (Figure 4B); however, rmC8 has a weaker propensity to obtain secondary structure in the presence of DPC. Changes in recombinant MBP conformation were, thus, induced by the interaction between the protein and the DPC detergent, as also seen before [47]. Trp fluorescence was also used to confirm binding to DPC micelles, and a saturation level of binding for both proteins was observed at 0.1% DPC (Figure 5B). Since only one Trp residue is present in MBP, the result indicates its environment changes upon micelle binding; most likely, it gets buried within the hydrophobic core of the micelle.


Charge isomers of myelin basic protein: structure and interactions with membranes, nucleotide analogues, and calmodulin.

Wang C, Neugebauer U, Bürck J, Myllykoski M, Baumgärtel P, Popp J, Kursula P - PLoS ONE (2011)

Features of MBP interaction with membranes and micelles.A. CD analysis of MBP conformation bound to lipid vesicles. Dotted line, MBP in phosphate buffer; solid line, MBP bound to DMPG/DMPC vesicles at a protein/lipid ratio of 1/300. MRE, mean residue ellipticity. B. The interaction with DPC micelles, as observed by intrinsic tryptophan fluorescence. Both rmC1 (black) and rmC8 (red) have increased Trp fluorescence in the presence (thick lines) of 0.1% DPC, compared to its absence (thin lines). The identical result was observed at temperatures between +25 and +42°C, and in the DPC concentration range 0.1–2.0%. The shift of the peak to a much lower wavelength while increasing its intensity is a sign that the single Trp residue of MBP is buried inside the DPC micelle, in a hydrophobic environment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102069&req=5

pone-0019915-g005: Features of MBP interaction with membranes and micelles.A. CD analysis of MBP conformation bound to lipid vesicles. Dotted line, MBP in phosphate buffer; solid line, MBP bound to DMPG/DMPC vesicles at a protein/lipid ratio of 1/300. MRE, mean residue ellipticity. B. The interaction with DPC micelles, as observed by intrinsic tryptophan fluorescence. Both rmC1 (black) and rmC8 (red) have increased Trp fluorescence in the presence (thick lines) of 0.1% DPC, compared to its absence (thin lines). The identical result was observed at temperatures between +25 and +42°C, and in the DPC concentration range 0.1–2.0%. The shift of the peak to a much lower wavelength while increasing its intensity is a sign that the single Trp residue of MBP is buried inside the DPC micelle, in a hydrophobic environment.
Mentions: The effects of several membrane mimics, including DPC and other phosphocholines, LDAO, SDS, and TFE, on the secondary structure of MBP were investigated by SRCD (Figure 4). It was pointed out that DPC micelles can constitute a realistic model of membrane interfaces [45], and thus, we decided to study the effect of different concentrations of DPC on the structure of rmMBP. Such experiments were carried out already previously with rmC1 and conventional CD spectroscopy [46], at much lower protein concentration. With the addition of DPC, rmC1 acquired a significant fraction of helical secondary structure (Figure 4A). In addition, all the three distinctive helical peaks increased with increasing DPC concentration from 25 mM to 100 mM. The effect of DPC on rmC8 is similar to rmC1 (Figure 4B); however, rmC8 has a weaker propensity to obtain secondary structure in the presence of DPC. Changes in recombinant MBP conformation were, thus, induced by the interaction between the protein and the DPC detergent, as also seen before [47]. Trp fluorescence was also used to confirm binding to DPC micelles, and a saturation level of binding for both proteins was observed at 0.1% DPC (Figure 5B). Since only one Trp residue is present in MBP, the result indicates its environment changes upon micelle binding; most likely, it gets buried within the hydrophobic core of the micelle.

Bottom Line: Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1.Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM.Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oulu, Oulu, Finland.

ABSTRACT
As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids--a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.

Show MeSH
Related in: MedlinePlus