Expression and properties of the recombinant murine Golli-myelin basic protein isoform J37.
A recombinant form of the murine Golli-myelin basic protein (MBP) isoform J37 (rmJ37) has been expressed in Escherichia coli and isolated to 95% purity via metal chelation and ion exchange chromatography. The protein did not aggregate lipid vesicles containing acidic phospholipids, unlike the 18.5 kDa isoform of MBP. This result is consistent with J37 having a functional role prior to the assembly of compact myelin. Circular dichroic spectroscopy showed that rmJ37 had a large proportion of random coil in aqueous solution but gained alpha-helix and beta-sheet in the presence of monosialoganglioside G(M1) and PI(4)P. Thus, like "classic" MBP, J37 is intrinsically unstructured, and its conformation depends on its environment and bound ligands. Analyses of the amino acid sequence of rmJ37 predicted an N-terminal calmodulin (CaM)-binding site. It was determined via a gel-shift assay and fluorescence spectroscopy that rmJ37 and CaM interacted in a 1:1 ratio in a Ca(2+)-dependent manner. However, the interaction was weak compared with 18.5 kDa MBP.
Kaur
Jaspreet
J
Department of Molecular Biology and Genetics, and Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada.
Libich
David S
DS
Campagnoni
Celia W
CW
Wood
D Denise
DD
Moscarello
Mario A
MA
Campagnoni
Anthony T
AT
Harauz
George
G
D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D051379:Mice; D004676:Myelin Basic Protein / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D002147:Calmodulin; C499047:Mbp protein, mouse; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014157:Transcription Factors