Terminal deletion mutants of myelin basic protein: new insights into self-association and phospholipid interactions.
The 18.5kDa isoform of myelin basic protein (MBP) has strong and probably specific interactions with phosphoinositides that are of interest regarding this protein's function, and in effecting its two-dimensional crystallization for structural determination. We have designed and constructed truncation mutants of recombinant 18.5kDa murine myelin basic protein (rmMBP) lacking either the N- or C-terminal third, i.e. rmMBPDeltaN and rmMBPDeltaC, respectively. Both variants rmMBPDeltaC and rmMBPDeltaN generally had a reduced ability to aggregate lipid vesicles, compared to the whole protein, especially at lower protein/lipid ratios. Lipid vesicle cosedimentation showed that both truncated variants exhibited altered binding with phosphatidylinositol (PI). Incubation of these proteins under monolayers comprising PI and a nickel-chelating lipid yielded crystalline arrays of rmMBPDeltaC (but not rmMBPDeltaN) in the absence of high salt or osmolytes, which are required for crystallization of whole protein. This result suggests that the C-terminal segment of MBP is a significant source of conformational heterogeneity, and its removal will facilitate future planar or three-dimensional crystallization attempts. Incubation of rmMBPDeltaN and rmMBPDeltaC under monolayers comprising phosphatidylinositol-4-phosphate and a nickel-chelating lipid yielded tubular structures of opposite chirality, suggesting a synergistic effect of both termini of MBP in organizing myelin lipids.
Hill
Christopher M D
CM
Department of Molecular Biology and Genetics, and Biophysics Interdepartmental Group, University of Guelph, Ont., Canada.
Haines
Jeffery D
JD
Antler
Christine E
CE
Bates
Ian R
IR
Libich
David S
DS
Harauz
George
G
D000595:Amino Acid Sequence; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D008055:Lipids / Q000737:chemistry*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D004676:Myelin Basic Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009186:Myelin Sheath / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D008055:Lipids; D004676:Myelin Basic Protein; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; C037178:phosphatidylinositol 4-phosphate; D009532:Nickel