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original natural myelin lipids, after removing the detergent (Riccio et al .,
2002). Myelin lipids were recovered by treatment of brain powder with
organic solvents according to the procedure of Deibler et al ., (1972), or by
removal of lipids from LB-MBP using a CHCl 3 /CH 3 OH 2:1 mixture.
Reconstitution of MBP into liposomes was achieved following
slightly different procedures, all based on the replacement of detergent
(removed) by myelin lipids (added). Classic, acid-extracted LF-MBP and
non-raft LB-MBP were studied. After the reconstitution procedure, SDS-
PAGE showed that both the LB-MBP and the LF-MBP remain intact.
Densitometric analysis of the lipids, extracted from both MBP forms and
fractionated by HPTLC, revealed that the reconstituted LB-MBP was
enriched in lipids with respect to the same protein in solution. In contrast,
LF-MBP was found to bind only a very low percentage of lipids.
Using electron microscopy and spectroscopic techniques, we have
investigated the structure of both lipid-free and lipid-bound MBP, after
reconstitution in liposomes made of myelin lipids, in order to verify the
differences in their interaction with liposomes and to evaluate the effects
on their structure of the reconstitution in a lipid microenvironment very
similar to the native one. The aim of this study was to understand not only
the kind of protein-lipid interaction, but also how much this interaction
affects the correct functional folding of the protein.
Electron microscopy showed that LF-MBP was adherent to the lipo-
some surface without penetrating the membrane. The CD measurements
carried out to investigate the secondary structure of LF-MBP (Table 1),
did not show relevant structural changes on LF-MBP in the presence of
myelin liposomes. In contrast, comparing the electron microscopy results
obtained on the LB-MBP in solution and in the presence of myelin lipo-
somes, the protein seemed to penetrate into liposomes. In addition, the CD
spectra showed a higher amount of ordered secondary structure than in the
LB-MBP with the detergent, indicating a transition to a higher content of
β
structure (Table 1). Interaction of LF-MBP or LB-MBP in liposomes
made of dimyristoyl-l
-helix
content in both proteins (not shown). A high percentage of random coil
was observed only in the lipid-free MBP.
α
-phosphatidic acid (DMPA) increased the
α
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