Purification and spectroscopic characterization of the recombinant BG21 isoform of murine golli myelin basic protein.
A recombinant form of the murine Golli-myelin basic protein (MBP) isoform BG21 (rmBG21) has been expressed in E. coli, and isolated to 96% purity via metal chelation chromatography. Characteristic yields were 6-8 mg protein per liter of culture in either minimal M9 or standard Luria-Bertani media. Circular dichroism spectroscopy showed that rmBG21 had a large proportion of random coil in aqueous solution, but gained alpha-helix in the presence of monosialoganglioside G(M1) and PI(4)P, as well as in the membrane-mimetic solvent trifluoroethanol. Bioinformatics analyses of the amino acid sequence of rmBG21 predicted an N-terminal calmodulin (CaM)-binding site. It was determined by fluorescence spectroscopy and dynamic light scattering that rmBG21 and CaM interacted weakly in a 1:1 ratio in a Ca(2+)-dependent manner. Solution NMR spectra of uniformly [(13)C(15)N]-labeled protein in aqueous buffer were consistent with it being an extended protein; spectral quality was independent of temperature. Thus, like "classic" MBP and the Golli-MBP isoform J37, rmBG21 is intrinsically disordered, implying multi functionality, and that its conformation depends on its environment and bound ligands.
Bamm
Vladimir V
VV
Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada.
Ahmed
Mumdooh A M
MA
Ladizhansky
Vladimir
V
Harauz
George
G
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
C499047:Mbp protein, mouse; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014157:Transcription Factors