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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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Related in: MedlinePlus

Size exclusion chromatography of MBP-CaM complexes at different stoichiometries.A. rmC1. B. rmC8. Black, MBP alone; red, CaM alone; blue, 1∶1 ratio (MBP∶CaM); green, 1∶2 ratio; orange, 1∶3 ratio; magenta, 1∶5 ratio; black dashed line, 2∶1 ratio. The elution volumes of calibration standards were as follows: 158 kDa, 12.90 ml; 75 kDa, 14.41 ml; 44 kDa, 15.20 ml; 29 kDa 16.61 ml; 13.7 kDa, 18.28 ml.
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pone-0019915-g007: Size exclusion chromatography of MBP-CaM complexes at different stoichiometries.A. rmC1. B. rmC8. Black, MBP alone; red, CaM alone; blue, 1∶1 ratio (MBP∶CaM); green, 1∶2 ratio; orange, 1∶3 ratio; magenta, 1∶5 ratio; black dashed line, 2∶1 ratio. The elution volumes of calibration standards were as follows: 158 kDa, 12.90 ml; 75 kDa, 14.41 ml; 44 kDa, 15.20 ml; 29 kDa 16.61 ml; 13.7 kDa, 18.28 ml.

Mentions: Size exclusion chromatography was used to analyze the complex formation between rmC1/rmC8 and CaM. Figure 7 shows the chromatograms at different rmMBP∶CaM ratios for rmC1 and rmC8. MBP and CaM alone elute, compared to regular globular markers, at apparent molecular weights of 29–33 kDa, reflecting their highly elongated structures, and thus, large hydrodynamic radii. The complexes with rmMBP∶CaM ratios of 1∶1 and 1∶2 obviously have different elution volumes, and there is no peak indicating the presence of uncomplexed CaM. However, the samples with rmMBP∶CaM ratios higher than 1∶2 have an elution volume close to that of the 1∶2 complex, and another separate peak appears, which exactly corresponds to CaM, not present in the complex. These results prove the formation of a stable 1∶2 complex between CaM and both rmC1and rmC8. The results also closely resemble those seen for brain MBP [8], indicating CaM complex formation by the recombinant forms of MBP is similar to proteins isolated from nervous tissue. Complex interactions and different oligomeric forms were also previously observed in gel shift assays [56]. An analysis of a 2∶1 mixture of MBP∶CaM surprisingly results in a new peak between the 1∶1 peak and the uncomplexed proteins, possibly resulting from a fast exchange between the complexed and monomeric forms. Overall, the data indicate that the MBP-CaM complex is heterogeneous, depending on the ratio of the binding partners; the different forms of the complex are stable enough to be resolved during chromatography.


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)

Size exclusion chromatography of MBP-CaM complexes at different stoichiometries.A. rmC1. B. rmC8. Black, MBP alone; red, CaM alone; blue, 1∶1 ratio (MBP∶CaM); green, 1∶2 ratio; orange, 1∶3 ratio; magenta, 1∶5 ratio; black dashed line, 2∶1 ratio. The elution volumes of calibration standards were as follows: 158 kDa, 12.90 ml; 75 kDa, 14.41 ml; 44 kDa, 15.20 ml; 29 kDa 16.61 ml; 13.7 kDa, 18.28 ml.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0019915-g007: Size exclusion chromatography of MBP-CaM complexes at different stoichiometries.A. rmC1. B. rmC8. Black, MBP alone; red, CaM alone; blue, 1∶1 ratio (MBP∶CaM); green, 1∶2 ratio; orange, 1∶3 ratio; magenta, 1∶5 ratio; black dashed line, 2∶1 ratio. The elution volumes of calibration standards were as follows: 158 kDa, 12.90 ml; 75 kDa, 14.41 ml; 44 kDa, 15.20 ml; 29 kDa 16.61 ml; 13.7 kDa, 18.28 ml.
Mentions: Size exclusion chromatography was used to analyze the complex formation between rmC1/rmC8 and CaM. Figure 7 shows the chromatograms at different rmMBP∶CaM ratios for rmC1 and rmC8. MBP and CaM alone elute, compared to regular globular markers, at apparent molecular weights of 29–33 kDa, reflecting their highly elongated structures, and thus, large hydrodynamic radii. The complexes with rmMBP∶CaM ratios of 1∶1 and 1∶2 obviously have different elution volumes, and there is no peak indicating the presence of uncomplexed CaM. However, the samples with rmMBP∶CaM ratios higher than 1∶2 have an elution volume close to that of the 1∶2 complex, and another separate peak appears, which exactly corresponds to CaM, not present in the complex. These results prove the formation of a stable 1∶2 complex between CaM and both rmC1and rmC8. The results also closely resemble those seen for brain MBP [8], indicating CaM complex formation by the recombinant forms of MBP is similar to proteins isolated from nervous tissue. Complex interactions and different oligomeric forms were also previously observed in gel shift assays [56]. An analysis of a 2∶1 mixture of MBP∶CaM surprisingly results in a new peak between the 1∶1 peak and the uncomplexed proteins, possibly resulting from a fast exchange between the complexed and monomeric forms. Overall, the data indicate that the MBP-CaM complex is heterogeneous, depending on the ratio of the binding partners; the different forms of the complex are stable enough to be resolved during chromatography.

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