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Measuring chemical activity of cholesterol𝝁= 𝝁𝟎+ 𝑹𝑻𝒍𝒏𝒂
Space-filling model of b-cyclodextrin
Cyclodextrin binds lipophilic drugs, including cholesterol
Regular Solution Theory
lclc
lBll
cBcc
xxNN
xTkN
xTkNF
ln
ln
ϴ is a parameter that accounts for interactions between components (i.e., it accounts for non-ideality), μc, μl are standard chemical potentials, and xc, xl are the mole fractions of cholesterol and the phospholipid.
0.0 0.1 0.2 0.3 0.4 0.5
0.2
0.4
0.6
0.8
DOPC
0.1 0.2 0.3 0.4 0.50.00
0.25
0.50
0.75
POPC
0.20 0.25 0.30 0.35 0.40 0.45 0.500.00
0.05
0.10
0.15
0.20
0.25
SM
0.1 0.2 0.3 0.4 0.50.0
0.1
0.2
0.3
0.4
0.5
0.6
DPPC
Cholesterol Concentration, c
Cholesterol Activity, a
Θ, kT S, %
DOPC 1.17 68
POPC -2.93 64
SM -6.27 85
DPPC -1.56 79
Θ: Interaction ParameterS: Maximum Cholesterol Solubility
Problems
• Develop theories that predict strength of pair-wise interactions from experimental activity-cholesterol relationships.
• Generalize mean field theories, such as RST, to account for limited solubilities.
)
Fluorescence microscopy of planar bilayer membranes
Objective
Solvent-containing Torus
Cholesterol/SM rafts form below the Tm of the SM
DOPC/DOPE (2:1), 15 mol % cholesterol and egg-SM (mostly 16:0,Tm = 41 °C)
Probe: 5 mol % rho-DOPE
Scale bar: 50 μm for A and B; 4 μm for C
Solid-gel domains are noncircular
A. 25 mol % DPPS (Tm = 55 °C) and 25 mol % cholesterolB. 25 mol % DMPE (Tm = 50 °C) and 25 mol % cholesterol
Scale bar: 4 μm
Rafts are fluid
Rafts are deformable
A. Pipette pulled 3-5 μm/s
B. Pipette withdrawn, 5 frames after A
C. Raft is circular, 9 frames after B
Scale bar: 50 μm
Lipids are more ordered in rafts than in a
surrounding membrane
raft
Rafts dissolve at temperatures above Tm of the SM
Times after raising temperature: A. 1 min, B. 5 min, C. 7 min, D. 9 min, E. 23 min
Membrane moved at D to show large dark domain (arrow). Large domains tendedto accumulate at the Gibbs-Plateau border
Question set # 1
•Why are rafts liquid-ordered rather than solid-ordered even though the bilayer is at a temperature below the Tm of the SM?
• Is the dominant effect the intercalation of cholesterol between the acyl chains that prevents the lipid tilting of the gel phase?
Raft formation requires saturated acyl chains and
depends on headgroupAcyl chain Domains with
cholesterol?
16:0 egg-SM Yes
18:0 SM or DSPC Yes
18:1 SM or DOPC No
18:0 DSPS No
14:0 DMPE No
16:0 and 18:0 GM1 No
Question Set # 2
•Why does either SM or PC phase separate with cholesterol?
• What are the relative roles of headgroup interactions and hydrogen bonding in raft formation? Are interactions between chains the primary determinant?
• Does SM or PC form stoichiometric complexes with cholesterol?
Rafts extend through both monolayers of a bilayer membrane
Torus
Question set # 3
•Why do SM/Cholesterol domains couple between the two monolayers?
• Is the cause interdigitation of the saturated acyl chains of SM of one monolayer with that of the other? Or do the side chains of cholesterol in the two monolayers interact?
• Are there other possibilities for coupling in a pure lipid bilayer?
Coupling of rafts in outer leaflets to lo domains of
inner leaflets
Question set # 4
•Why are rafts small in biological cell membranes but large in lipid bilayer membranes?
• Do membrane proteins serve as nucleating centers for raft formation, rather than passively partition into rafts?
Surface vs. cross-section observation of rafts
cross-section
rafts in 3D
NBD-DPPE
Rho-DOPE
GUV
raft
Composition:SM, Cholesterol, DOPC
Large raft formation as a function of cholesterol content
10% Cholesterol 40% Cholesterol
20% Cholesterol 20% SM20% SM20% SM
40% DOPC60% DOPC70% DOPC
Increasing cholesterol in a SM/DOPC GUV
0 sec
200 sec160 sec120 sec
80 sec
40 sec
Add methyl beta-cyclodextrin/cholesterol
Increasing Cholesterol
10 15 20 25 30 35 40 45 50 55
1.00
1.05
1.10
1.15
1.20
Flu
ore
sce
nce
(u
nq
ue
nch
ed
/qu
en
che
d)
T, oC
0.1% NBD-DPPE 1% Rho-DOPE
Small rafts are present at high cholesterol content in the absence of tension
Question Set # 5
Why can’t large rafts exist at high Cholesterol content?
Why don’t small rafts merge at high cholesterolcontent?
0 sec
100 sec80 sec60 sec
40 sec
20 sec
Tension promotes large rafts
20% Sphingomyelin, 40% cholesterol
Large rafts appear after liposome swelling
Swelling induces large rafts in cells at 37oC
unswollen
swollen
Cholera Toxin B – marker for GM1
OuabainCholera Toxin B
Ouabain – marker for Na/K-ATPase
in entropy
RnrN
nnkTF 22ln min
B
Raft merger is a competition between a reduction
in boundary energy and a decrease in entropy
Rafts are thicker than the surround
raft
Lateral tension increases line tension
of rafts
A
dAk
JB
JB
F
220
20 22
div2
tn
Question # 6
•Does mechanical tension increase line tension of domains that have the same thickness as the surround?
Which one is Artem?