Amphotericin B induced interdigitation of apolipoprotein stabilized nanodisk bilayers Page: 4 of 12
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infected with either Candida albicans (1) or
the protozoal parasite, Leishmania major (2)
revealed that AMB-ND elicit strong antibiotic
effects. Recent studies investigating the
unique spectroscopic properties of this
heptaene macrolide antibiotic in ND revealed
AMB concentration and temperature
dependent changes in spectral properties
consistent with AMB self association in the
ND lipid milieu (3).
A question that arises relates to
whether AMB distributes among ND particles
in a uniform manner or selectively partitions
to create a population of AMB rich particles
that co-exist with particles lacking AMB. At
ratios of phospholipid:AMB used in the study
by Hargreaves et al. (2006) (4:1, 40:1 or 400:1
w/w), a uniform distribution model would
require that AMB molecules be individually
surrounded by phospholipid. Given the
amphoteric nature of AMB and its propensity
to self-associate (4), such a model seems
unlikely.
In an effort to better define the
molecular organization of AMB, phospholipid
and apolipoprotein in AMB-ND, gel
permeation chromatography, electrophoresis
and atomic force microscopy studies have
been conducted. The data obtained indicate
that, at high phospholipid:AMB ratios, AMB
does not partition uniformly among ND
particles but, rather, combines with
phospholipid at an optimal ratio to create a
subpopulation of apolipoprotein stabilized
particles that possess a ~1:1
phospholipid:AMB molar ratio. Increasing
the starting AMB content to achieve a 1:1
phospholipid:AMB molar ratio results in
generation of a uniform population of larger
diameter ND in which the bilayer is
interdigitated.
EXPERIMENTAL PROCEDURES
Materials. AMB (USP grade) was
obtained from Research Organics Inc.Dimyristoylphosphatidylcholine (DMPC) was
from Avanti Polar Lipids Inc. Recombinant
apolipoprotein A-I (apoA-I) was produced as
previously described (5).
AMB-ND preparation. Phospholipid vesicle
dispersions were prepared as described earlier
(1). To the dispersed lipid a given amount of
AMVIB from a stock solution (30 mg/ml in
dimethylsulfoxide; DMSO) was added in a
subdued light environment. Subsequent
addition of apoA-I in buffer leads to a time-
dependent decrease in sample turbidity with
full sample clarity achieved by bath sonication
with the sample temperature maintained below
25 0C. AMB-ND preparations contained 0,
2.5 or 12 mg AMB per 10 mg DMPC and 4
mg apolipoprotein. In the latter case, this
corresponds to slightly higher than a 1:1
phospholipid:AMB molar ratio. When higher
amounts of AMVIB were introduced to the
reaction mix, precipitation occurred with loss
of AMB. All preparations were dialyzed
overnight at 4 0C and filter sterilized (0.22
pm) before use.
Gelfiltration chromatography. Samples were
applied to a 2.5 x 40 cm column of Sepharose
6B equilibrated in phosphate buffered saline
(0.1 M sodium phosphate, pH 7.0, 150 mM
sodium chloride) and eluted in 2 ml fractions.
Far UV circular dischroism spectroscopy. Far
UV circular dichroism (CD) measurements
were performed on a Jasco 810
spectropolarimeter. Protein samples were
dissolved in 50 mM sodium phosphate, pH
7.0. For guanidine HCl denaturation
experiments, samples (0.2 mg/mL) were
incubated overnight at a given denaturant
concentration in order to attain equilibrium,
and ellipticity was measured at 222 nm (0.1
cm path length). Ellipticity values were
converted into molar ellipticity (millidegrees
cm2 decimole-) using a mean residue weight
value of 113.4 for wild type and 114.3 for the
deletion mutant. Protein secondary structure
content was calculated using the self-consisted
method with Dicroprot version 2.6 (6).2
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Nguyen, T; Weers, P M; Sulchek, T; Hoeprich, P D & Ryan, R O. Amphotericin B induced interdigitation of apolipoprotein stabilized nanodisk bilayers, article, December 7, 2006; Livermore, California. (https://digital.library.unt.edu/ark:/67531/metadc894885/m1/4/: accessed April 23, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.