Supporting information
Kinetic and Thermodynamic Analysis of Cholesterol
Transfer between Phospholipid Vesicles and Nanodiscs
Naoya Matsuzaki1, Tetsurou Handa2, and Minoru Nakano3,*
1
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
2
Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3
Minami-Tamagaki-cho, Suzuka, Mie, 513-8670, Japan
3
Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani,
Toyama 930-0194, Japan
S1
Derivation of equation (1)
Scheme of the Chol transfer between LUV and nanodisc.
The changes in Chol concentration in nanodiscs and in outer and inner leaflets of LUVs are
expressed by equations (S1) – (S3), respectively.
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−
d [Chol(t)] ND
−
d [Chol(t)] out
−
d [Chol(t)] in
dt
(S1)
= kLUV [Chol(t)] out ⋅ PND – kND [Chol(t)] ND ⋅ PLUV + kf [Chol(t)] out – kf [Chol(t)] in (S2)
dt
dt
= kND [Chol(t)] ND ⋅ PLUV – kLUV [Chol(t)] out ⋅ PND
= kf [Chol(t)] in – kf [Chol(t)] out
(S3)
where [Chol(t)] out and [Chol(t)] in are the concentrations (i.e., molarities in solution (mol/L), but
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not the mole fractions in the membrane) of Chol located at outer and inner leaflets of LUVs,
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respectively, and thus [Chol(t)] LUV = [Chol(t)] in + [Chol(t)] out .
PND and PLUV are the probability of
Chol to be incorporated from outer aqueous media into nanodiscs and LUVs, respectively, and are
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determined by the concentration of PLs facing to the outer aqueous media as follows:
PND =
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[ PL] ND
[ PL] ND + [ PL] LUV
PLUV =
[ PL] LUV 2
[ PL] ND + [ PL] LUV
(S4)
2
(S5)
2
S2
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Flip-flop of Chol is known to be extremely fast (Steck, T. L. et al. Biophys. J. 2002, 83, 2118;
Hamilton, J. A. Curr. Opin. Lipidol. 2003, 14, 263).
Under this condition Chol concentration in the
inner leaflet of LUVs immediately equilibrates with that of the outer leaflet, and thereby
− d [Chol(t)] in dt in equation (S3) equals to 0.
Accordingly,
1
[Chol(t)] in = [Chol(t)] out = 2 [Chol(t)] LUV
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(S6)
From equations (S1) – (S3), the Chol concentration changes for nanodiscs and LUVs are finally
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given as follows:
−
d [Chol(t)] ND
−
d [Chol(t)] LUV
dt
dt
1
= kND [Chol(t)] ND ⋅ PLUV – kLUV [Chol(t)] LUV ⋅ PND
2
=−
d [Chol(t)] out
−
(S7)
d [Chol(t)] in
dt
dt
1
= kLUV [Chol(t)] LUV ⋅ PND – kND [Chol(t)] ND ⋅ PLUV
2
Equation (S7) corresponds to equation (1) in the main text.
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S3
(S8)
Figure S1.
The gel filtration chromatography profile of a mixture of LUVs and nanodiscs.
LUVs
(PC/Chol = 100/19, 500 µM PC) and PC nanodiscs (500 µM PC) were incubated at 37°C for 24 h
and subsequently loaded into a Sepharose CL-6B column.
The elution profile was monitored at the
excitation/emission wavelengths of 385 nm/460 nm, detecting fluorescence of 2-AS incorporated
into particles.
Peaks at ca. 1,200 s and 2,300 s correspond to LUV and nanodisc, respectively.
S4
Figure S2.
Calcein leakage from LUVs.
Calcein-encapsulating LUVs were prepared with Tris
buffer (10 mM Tris-HCl, 0.01% NaN3, pH 7.4) containing 70 mM calcein and were loaded into a
Sepharose CL-6B column to remove calcein that was not encapsulated.
Calcein-encapsulating
LUVs (10 µM PC) and calcein-free LUVs (90 µM PC) were mixed together with nanodiscs
(PC/Chol = 100:0 (●) or 100/12 (▲)) or lipid-free apoA-I (■) at 37°C, to a final apoA-I
concentration of 0.5 µM, and the changes in the fluorescence intensity were monitored at the
excitation/emission wavelengths of 490 nm/520 nm.
Calcein leakage was calculated by 100 × (F –
F0) ⁄ (F100 – F0), where F0 and F are the fluorescence intensity before and after adding nanodiscs (or
apoA-I), respectively, and F100 is the fluorescence intensity after adding 0.3% Triton X-100.
S5