title: Substitutions mimicking deimination and phosphorylation of 18.5-kDa myelin basic protein exert local structural effects that subtly influence its global folding.
abstract: Intrinsically-disordered proteins (IDPs) present a complex interplay of conformational variability and multifunctionality, modulated by environment and post-translational modifications. The 18.5-kDa myelin basic protein (MBP) is essential to the formation of the myelin sheath of the central nervous system and is exemplary in this regard. We have recently demonstrated that the unmodified MBP-C1 component undergoes co-operative global conformational changes in increasing concentrations of trifluoroethanol, emulating the decreasing dielectric environment that the protein encounters upon adsorption to the oligodendrocyte membrane [K.A. Vassall et al., Journal of Molecular Biology, 427, 1977-1992, 2015]. Here, we extended this study to the pseudo-deiminated MBP-C8 charge component, one found in greater proportion in developing myelin and in multiple sclerosis. A similar tri-conformational distribution as for MBP-C1 was observed with slight differences in Gibbs free energy. A more dramatic difference was observed by cathepsin D digestion of the protein in both aqueous and membrane environments, which showed significantly greater accessibility of the F42-F43 cut site of MBP-C8, indicative of a global conformational change. In contrast, this modification caused little change in the protein's density of packing on myelin-mimetic membranes as ascertained by double electron-electron resonance spectroscopy [D.R. Kattnig et al., Biochimica et Biophysica Acta (Biomembranes), 1818, 2636-2647, 2012], or in its affinity for Ca(2+)-CaM. Site-specific threonyl pseudo-phosphorylation at residues T92 and/or T95 did not appreciably affect any of the thermodynamic mechanisms of conformational transitions, susceptibility to cathepsin D, or affinity for Ca(2+)-CaM, despite previously having been shown to affect local structure and disposition on the membrane surface.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Kenrick A
authors.initials: KA
authors.lastname: Vassall
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Bamm
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Jenkins
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada; Max-Planck-Institut für Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany.
authors.forename: Caroline J
authors.initials: CJ
authors.lastname: Velte
authors.affiliation: Max-Planck-Institut für Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Kattnig
authors.affiliation: Molecular Structure and Function Program, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada.
authors.forename: Joan M
authors.initials: JM
authors.lastname: Boggs
authors.affiliation: Max-Planck-Institut für Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany.
authors.forename: Dariush
authors.initials: D
authors.lastname: Hinderberger
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada. Electronic address: gharauz@uoguelph.ca.
authors.forename: George
authors.initials: G
authors.lastname: Harauz
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D007097:Imines / Q000378:metabolism*; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017510:Protein Folding; D059748:Proteolysis; D013050:Spectrometry, Fluorescence; D053835:Unilamellar Liposomes
chemical_list: D007097:Imines; D004676:Myelin Basic Protein; D053835:Unilamellar Liposomes
keywords: Amphipathic α-helix; Calmodulin; Cathepsin D; Circular dichroism (CD) spectroscopy; Citrullination/deimination; DEER spectroscopy; Förster (fluorescence) resonance energy transfer (FRET); Intrinsically-disordered protein; Molecular recognition fragment (MoRF); Myelin basic protein (MBP); Phosphorylation; Protein folding intermediate