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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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SPR analysis of MBP binding to lipidic mono- and bilayers.Black, PC; red, PC-PI; blue, PC-PIP; green, PC-PIP2. Curve fitting was carried out with a sigmoidal dose-response model. A. rmC1 on lipid monolayers. B. rmC8 on lipid monolayers. C. rmC1 on lipid bilayers. D. rmC8 on lipid bilayers.
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pone-0019915-g006: SPR analysis of MBP binding to lipidic mono- and bilayers.Black, PC; red, PC-PI; blue, PC-PIP; green, PC-PIP2. Curve fitting was carried out with a sigmoidal dose-response model. A. rmC1 on lipid monolayers. B. rmC8 on lipid monolayers. C. rmC1 on lipid bilayers. D. rmC8 on lipid bilayers.

Mentions: Interactions between rmMBP and supported lipids with different compositions as a monolayer were investigated using SPR (Figure 6A,B). While rmC1 and rmC8 bind the monolayers in a similar manner, the affinity and total binding capacity are both slightly lower for the rmC8 isoform. The binding also shows a cooperative nature, with signal being detectable only above 0.1 µM MBP concentration, and the binding is saturated at approximately 0.3 µM protein concentration for both rmC1 and rmC8; the exception is rmC8 binding to phosphatidylcholine, where saturation only occurs at 0.5 µM (Figure 6B). With increasingly negative membrane surface charge, the binding of rmMBP increases significantly. This suggests that the interaction between MBP and lipid is largely electrostatic, and that the binding density of MBP on the lipid surface increases with negative charge in the lipid headgroups. In line with these data, it has been shown before that MBP binds negatively charged lipids with high affinity [51], [52], and that the binding can be very tight [53].


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)

SPR analysis of MBP binding to lipidic mono- and bilayers.Black, PC; red, PC-PI; blue, PC-PIP; green, PC-PIP2. Curve fitting was carried out with a sigmoidal dose-response model. A. rmC1 on lipid monolayers. B. rmC8 on lipid monolayers. C. rmC1 on lipid bilayers. D. rmC8 on lipid bilayers.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102069&req=5

pone-0019915-g006: SPR analysis of MBP binding to lipidic mono- and bilayers.Black, PC; red, PC-PI; blue, PC-PIP; green, PC-PIP2. Curve fitting was carried out with a sigmoidal dose-response model. A. rmC1 on lipid monolayers. B. rmC8 on lipid monolayers. C. rmC1 on lipid bilayers. D. rmC8 on lipid bilayers.
Mentions: Interactions between rmMBP and supported lipids with different compositions as a monolayer were investigated using SPR (Figure 6A,B). While rmC1 and rmC8 bind the monolayers in a similar manner, the affinity and total binding capacity are both slightly lower for the rmC8 isoform. The binding also shows a cooperative nature, with signal being detectable only above 0.1 µM MBP concentration, and the binding is saturated at approximately 0.3 µM protein concentration for both rmC1 and rmC8; the exception is rmC8 binding to phosphatidylcholine, where saturation only occurs at 0.5 µM (Figure 6B). With increasingly negative membrane surface charge, the binding of rmMBP increases significantly. This suggests that the interaction between MBP and lipid is largely electrostatic, and that the binding density of MBP on the lipid surface increases with negative charge in the lipid headgroups. In line with these data, it has been shown before that MBP binds negatively charged lipids with high affinity [51], [52], and that the binding can be very tight [53].

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