Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments.
Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein-lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.
Zhong
Ligang
L
Department of Physics, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1.
Bamm
Vladimir V
VV
Ahmed
Mumdooh A M
MA
Harauz
George
G
Ladizhansky
Vladimir
V
D002247:Carbon Isotopes; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000737:chemistry*; D009587:Nitrogen Isotopes
D002247:Carbon Isotopes; D008051:Lipid Bilayers; D004676:Myelin Basic Protein; D009587:Nitrogen Isotopes