Membrane Formation, Micelles, Sterols,
Diffusion in Membranes, and Membrane Fusion
Paul H. Schlesinger
Department of Cell Biology and Physiology
paul@cellbiology.wustl.edu
362-2223
December 8, 2011
2011-12-08
Biology 5325: Stability,
Environment and Interaction
Membranes 5325
Biology 5325: Stability,
Environment and Interaction
Membrane Formation, Micelles, Sterols,
Diffusion in Membranes, and Membrane Fusion
Paul H. Schlesinger
Department of Cell Biology and Physiology
paul@cellbiology.wustl.edu
362-2223
December 8, 2011
Distinct from biological macromolecules membranes
are formed by assembling monomers using
nonconvalent bonds.
2011-12-08
Membrane Assembly
Membranes 5325
Basis for Membrane Stability
Hydrophobic Tanford
Membrane Assembly
Membrane Assembly
Distinct from biological macromolecules membranes
are formed by assembling monomers using
nonconvalent bonds.
2011-12-08
Membrane Assembly
Membranes 5325
Basis for Membrane Stability
Membrane Assembly
Hydrophobic Tanford
Membrane Assembly
Aqueous Amphiphile Solutions
• First noted that osmotic activity was less than
concentration and used the term micelle to describe the
small aggregates, McBain (1913).
• These concepts were extended and size and shape of the
concentration dependant particles was studied by Debye
during the first half of the 20th century.
• Osmotic Pressure ∝ monomers + micelles
• Turbidity is ∝ micelles and their size
Aqueous Amphiphile Solutions
• Surface Tension falls in propoetion to the soluble
monomer
Displacement from self
Molecular Dimensions of This Effect
2011-12-08
Amphipile Species
Membranes 5325
Basis for Membrane Stability
Amphipile Species
Molecular Dimensions of This Effect
Hydrophobic Tanford
Amphipile Species
• Bilayer formation and shape is driven by the molecular
geometry and dimensions providing curvature to long
chain lipid molecules.
• Stabilization of non-bilayer structures
– Minimal “detergent” stabilization forms bicelles by
the addition of a shorter chain amphiphile to cover
the hydrophobic regions of the aggregate.
– Phospholipid stabilization of non-anisotropic
amphiphiles produces fat droplets in cells
– Generalize to the effect of detegerts on membranes
and the formation of micelle containing proteins
Energy Required to Increase the Membrane Surface
dW = γdA
2011-12-08
Surface/Interfacial Tension
Membranes 5325
Basis for Membrane Stability
Interfacial Tension in Micelle Stability
Surface/Interfacial Tension
Surface/Interfacial Tension
Energy Required to Increase the Membrane Surface
dW = γdA
2011-12-08
Surface/Interfacial Tension
Energy Required to Increase the Membrane Surface
dW = γdA
• Therefore at constant T or P
γ=
∂G
∂A
(1)
T,V,n
• Spontaneous changes drive decreasing surface
area, spheres and larger particles
Membranes 5325
Basis for Membrane Stability
Interfacial Tension in Micelle Stability
Surface/Interfacial Tension
Energy Required to Increase the Membrane Surface
dW = γdA
• Therefore at constant T or P
Surface/Interfacial Tension
γ=
∂G
∂A
(1)
T,V,n
• Spontaneous changes drive decreasing surface
area, spheres and larger particles
from [?]
2011-12-08
Curvature Development
∆p = γ
1
1
+
Rx Ry
Membranes 5325
Basis for Membrane Stability
Curvature Development
∆p = γ
1
1
+
Rx Ry
Introduce Curvature as a Reflection of Energy
Curvature Development
(2)
• Surface tension - expand to include more that one
amphiphile, “Detergent”, that permites stabilization of the
first amphiphile.
• The two immiscible compounds must the negotiate
surface configuartion
1
1
• ∆p = γ Rx + Ry
• ∆p 0.01 (1mm) 1.436(1 µ) 143.6(10 nm) ATM
(2)
2011-12-08
Curvature Development
∆p = γ
1
1
+
Rx Ry
Membranes 5325
Basis for Membrane Stability
Introduce Curvature as a Reflection of Energy
Curvature Development
(2)
This requires that the pressure normal to the surface
is dramatically dependent upon the curvature.
wiki surface tension and curvature
Curvature Development
∆p = γ
1
1
+
Rx Ry
(2)
This requires that the pressure normal to the surface
is dramatically dependent upon the curvature.
2011-12-08
Membrane Curvature
Membranes 5325
SAXs: Measurement of Membrane Curvature on a nm Length
Scale
Membrane Curvature
Membrane Curvature
Addresses the measurement of structures in the 1000 t0 1 nm
length domain of hydrated samples.
Small Angle X-ray Scattering
2011-12-08
Membrane Curvature
Membranes 5325
SAXs: Measurement of Membrane Curvature on a nm Length
Scale
Membrane Curvature
Membrane Curvature
Small Angle X-ray Scattering
Analysis of Saxs Data
• Θ Incident–Scatter Angle
• s = 4π sin Θ/λ
• Solvent subtraction
2011-12-08
Membrane Curvature
Membranes 5325
SAXs: Measurement of Membrane Curvature on a nm Length
Scale
Membrane Curvature
Membrane Curvature
Analysis of Saxs Data
• Θ Incident–Scatter Angle
• s = 4π sin Θ/λ
• Solvent subtraction
Basis for Structural Interpretation
• Increased detector discrimination allows the measurement
of intensity in what used to be the saturated region close
to the incident beam.
• The scattering vector describes intensity as a function of
the angle and analysis at small angles is very dependent
upon the beam wavelength.
• The variation in the electron density occurs in the sample
at the interface but it allows study of the geometry of
these interfaces.
2011-12-08
Membrane Curvature
Membranes 5325
SAXs: Measurement of Membrane Curvature on a nm Length
Scale
Membrane Curvature
Membrane Curvature
2011-12-08
Membrane Curvature
Membranes 5325
SAXs: Measurement of Membrane Curvature on a nm Length
Scale
Membrane Curvature
Membrane Curvature
• Sterols in Membranes
H3C
CH3
CH3
H
H
H
HO
•
•
•
Profound modulation of membrane properties
Question of heterogenous phase structure
Sterol protein interaction
2011-12-08
Sterols
Membranes 5325
Sterols
Cholesterol
Sterols
Sterols
• Sterols in Membranes
H3C
CH3
CH3
H
H
H
HO
•
•
•
Profound modulation of membrane properties
Question of heterogenous phase structure
Sterol protein interaction
2011-12-08
Sterols
• Sterol Selection
Mammals
required
synthesized
taken up
M. mycoides
required
not synthesized
S. cerevesiae
not used
not synthesized
not used
-
-
required
synthesized
not used
not synthesized
not used
not synthesized
taken up
synthesized
Membranes 5325
Sterols
Cholesterol
Sterols
Sterols
• Sterol Selection
Mammals
required
synthesized
taken up
M. mycoides
required
not synthesized
S. cerevesiae
not used
not synthesized
not used
-
-
required
synthesized
not used
not synthesized
not used
not synthesized
taken up
synthesized
• Cholesterol Dependent Processes
• Transport e.g. Na+ ,K+ -ATPase
• Membrane Receptors e.g. SCAP
Direct Sterol-Protein Interaction should be
sensitive to depletion and subsitution
2011-12-08
Sterols
Membranes 5325
Sterols
Cholesterol
Sterols
Sterols
• Cholesterol Dependent Processes
• Transport e.g. Na+ ,K+ -ATPase
• Membrane Receptors e.g. SCAP
Direct Sterol-Protein Interaction should be
sensitive to depletion and subsitution
• However depletion will change membrane
properties and the effectiveness of rescue by
alternative sterols is not clear.
2011-12-08
Sterols
Membranes 5325
Sterols
Cholesterol
Sterols
Sterols
• However depletion will change membrane
properties and the effectiveness of rescue by
alternative sterols is not clear.
• Enantiomer Recognition
Condensing effect on membranes is identical
with the nat-cholesterol
2011-12-08
Sterols
Membranes 5325
Sterols
Cholesterol
Sterols
Sterols
• Enantiomer Recognition
Condensing effect on membranes is identical
with the nat-cholesterol
Diastereomers have the same chemical composition but they
differ in relative chemical configuration at different portions of
the molecule.
• Specific types of interactions
• Organisms–c. elegans vs mammals
• Proteins–individual cases
• Membranes ????
E. J. Westover and D. F. Covey (2004). The Enantiomer
of Cholesterol. J. Memb. Biol. 202:61-72.
2011-12-08
Sterols
Membranes 5325
Sterols
Cholesterol
Sterols
Sterols
• Specific types of interactions
• Organisms–c. elegans vs mammals
• Proteins–individual cases
• Membranes ????
E. J. Westover and D. F. Covey (2004). The Enantiomer
of Cholesterol. J. Memb. Biol. 202:61-72.
• Cholesterol in Membranes
•
Cholesterol is amphipathic
•
3β -OH
•
Ring structure packs with the
alkane chains
•
This orders and condenses
membranes
•
Inhibits the formation of
crystalline/gel phase
2011-12-08
Sterols
Membranes 5325
Sterols
Cholesterol
Sterols
Sterols
• Cholesterol in Membranes
•
Cholesterol is amphipathic
•
3β -OH
•
Ring structure packs with the
alkane chains
•
This orders and condenses
membranes
•
Inhibits the formation of
crystalline/gel phase
• Phospholipid–Cholesterol Interaction
2011-12-08
Sterols
Membranes 5325
Sterols
Cholesterol
Sterols
Sterols
• Phospholipid–Cholesterol Interaction
• Langmuir trough, fluorescence dye, adjust pressure
• But no physical property changes are detected
• alakoskela:2008
2011-12-08
What if we...
Membranes 5325
Sterols
Oxysterols
25
21
23
18
19
1
2
3
11
13
6
26
B
25
21
23
18
12
19
1
2
3
11
4
15
8
7
5
6
16
14
9
10
20 22
17
13
24
27
24
16
15
8
7
5
26
B
25
21
13
9
14
7
6
• gale:2008 - Ory on Friday
16
15
8
5
20 22
17
11
10
4
27
23
18
2
3
• Important roles in regulation
7
5
4
14
12
15
8
13
9
10
19
• Naturally occurring
16
14
9
10
17
2
3
1
20 22
12
25
23
20 22
17
11
6
What if we...
• Alter intrinsic sterol dipole
24
26
21
18
12
19
1
4
26
A
What if we...
A
24
27
27
2011-12-08
What if we...
Membranes 5325
Sterols
Oxysterols
What if we...
What if we...
• z-membrane normal; xy-plane of the membrane
• β tilt deviation of sterol axis from membrane normal
• γ twist the deviation of sterol xy-plane from membrane
xy-plane
b
Ring Twist Angle (degrees)
180
150
120
90
60
30
0
−30
−60
−90
−120
−150
−180
0
a
30
60
90 120 150
Ring Tilt Angle (degrees)
180
2.5
180
2
1.5
1
a
b
0.5
0
0
30
60
90 120 150
Ring Tilt Angle (degrees)
180
150
120
90
60
30 b
0
0
a
30
60
90 120 150
Ring Tilt Angle (degrees)
180
Ring Twist Angle (degrees)
Ring Twist Angle (degrees)
b
a
30
60
90 120 150
Ring Tilt Angle (degrees)
180
180
b
150
120
90
60
30
0
−30
−60
−90
a
−120
−150
−180
0
0.5
1
1.5
2
2.5
Distance From Ring to Bilayer Center (nm)
2.5
180
Tail Tilt Angle (degrees)
What if we...
What if we...
180
150
120
90
60
30
0
−30
−60
−90
−120
−150
−180
0
Distance From Ring to Bilayer Center (nm)
2011-12-08
180
b
150
120
90
60
30
0
−30
−60
−90
a
−120
−150
−180
0
0.5
1
1.5
2
2.5
Distance From Ring to Bilayer Center (nm)
Tail Tilt Angle (degrees)
Distance From Ring to Bilayer Center (nm)
Ring Twist Angle (degrees)
What if we...
Membranes 5325
Sterols
Oxysterols
2
1.5
1
a
b
0.5
0
0
30
60
90 120 150
Ring Tilt Angle (degrees)
180
150
120
90
60
30 b
0
0
a
30
60
90 120 150
Ring Tilt Angle (degrees)
180
Ring Twist Angle (degrees)
180
150
120
90
60
30
0
−30
−60
−90
−120
−150
−180
0
b
f
a
d
c
e
30
60
90 120 150
Ring Tilt Angle (degrees)
180
2.5
180
2
1.5
c
b
a
1
0.5
0
0
d
e
f
30
60
90 120 150
Ring Tilt Angle (degrees)
180
f
150
120
d
90
b
e
a
60
30
0
0
c
30
60
90 120 150
Ring Tilt Angle (degrees)
180
Ring Twist Angle (degrees)
Ring Twist Angle (degrees)
b
f
a
d
c
e
30
60
90 120 150
Ring Tilt Angle (degrees)
180
180
150
b
120
90
f
60
30
a
0
d
−30
−60
−90
c
−120
e
−150
−180
0
0.5
1
1.5
2
2.5
Distance From Ring to Bilayer Center (nm)
2.5
180
2
1.5
c
Tail Tilt Angle (degrees)
What if we...
What if we...
180
150
120
90
60
30
0
−30
−60
−90
−120
−150
−180
0
Distance From Ring to Bilayer Center (nm)
2011-12-08
180
150
b
120
90
f
60
30
a
0
d
−30
−60
−90
c
−120
e
−150
−180
0
0.5
1
1.5
2
2.5
Distance From Ring to Bilayer Center (nm)
Tail Tilt Angle (degrees)
Distance From Ring to Bilayer Center (nm)
Ring Twist Angle (degrees)
What if we...
Membranes 5325
Sterols
Oxysterols
b
a
1
0.5
0
0
d
e
f
30
60
90 120 150
Ring Tilt Angle (degrees)
180
f
150
120
d
90
b
e
a
60
30
0
0
c
30
60
90 120 150
Ring Tilt Angle (degrees)
180
A
B
C
D
E
F
2011-12-08
What if we...
Membranes 5325
Sterols
Oxysterols
What if we...
What if we...
A
B
C
D
E
F
• Membrane Microdomains
•
Originally a concept, annular
lipids
•
Detergent resistant membranes
(DRMs)
•
Enriched in sphingolipids and
cholesterol
•
Atomistic to continuum model
•
Diffusion in Membranes
P.G. Saffman and M. Delbrük (1975). Brownian motion in biological membranes Proc. Nat. Acad. Sci USA
72:3111-3113.
2011-12-08
Lateral Anisotropy in Membranes
Membranes 5325
Diffusion in the Membrane
Lateral Anisotropy in Membranes
Lateral Anisotropy in Membranes
• Membrane Microdomains
•
Originally a concept, annular
lipids
•
Detergent resistant membranes
(DRMs)
•
Enriched in sphingolipids and
cholesterol
•
Atomistic to continuum model
•
Diffusion in Membranes
P.G. Saffman and M. Delbrük (1975). Brownian motion in biological membranes Proc. Nat. Acad. Sci USA
72:3111-3113.
• Diffusion in the membrane plane
• Einstein–Smoluchowski Equation
D=
•
kT
ξ
(3)
•
Jackson(2006) chapter 6
(5)
(3)
(4)
Stoke’s–Einstein Law
D=
• ξ frictional coefficient
kT
ξ
Stoke’s Law
ξ = 6πηa
•
(4)
Stoke’s–Einstein Law
kT
6πηa
• Diffusion in the membrane plane
• Einstein–Smoluchowski Equation
D=
Lateral Anisotropy in Membranes
• Diffusion and viscosity
D=
Lateral Anisotropy in Membranes
• Einstein established the relationship of diffusion and
friction
Stoke’s Law
ξ = 6πηa
•
2011-12-08
Lateral Anisotropy in Membranes
Membranes 5325
Diffusion in the Membrane
kT
6πηa
(5)
• Giant liposomes and diffusion
DMPC/cholesterol 1:1 +
DiI-C18
N. Kahya & P Schwille J. Fluoresc (2006) 16:671-8
2011-12-08
Lateral Anisotropy in Membranes
Membranes 5325
Diffusion in the Membrane
Lateral Anisotropy in Membranes
• Giant liposomes and diffusion
DMPC/cholesterol 1:1 +
DiI-C18
Lateral Anisotropy in Membranes
N. Kahya & P Schwille J. Fluoresc (2006) 16:671-8
• Diffusion of lipids in anisotropic membranes
A-DOPC/cholesterol •
A-SM/cholesterol 4
B-DOPC/cholesterol
C-SM enriched Lo 4
C-DOPC enriched Ld C-grey one phase
D-DOPC/SM/Cholesterol
1:1:1
2011-12-08
Lateral Anisotropy in Membranes
Membranes 5325
Diffusion in the Membrane
Lateral Anisotropy in Membranes
Lateral Anisotropy in Membranes
• Diffusion of lipids in anisotropic membranes
A-DOPC/cholesterol •
A-SM/cholesterol 4
B-DOPC/cholesterol
C-SM enriched Lo 4
C-DOPC enriched Ld C-grey one phase
D-DOPC/SM/Cholesterol
1:1:1
Singer & Nicolson (1972) Science 175:720-731
2011-12-08
Membrane Proteins
Membranes 5325
Diffusion in the Membrane
Inclusions
Membrane Proteins
Membrane Proteins
Singer & Nicolson (1972) Science 175:720-731
Singer & Nicolson (1972) Science 175:720-731
Proteins can be bound to membranes, integral
membrane protein, attached by a covalent lipid or
bound to another membrane protein
2011-12-08
Membrane Proteins
Membranes 5325
Diffusion in the Membrane
Inclusions
Membrane Proteins
Membrane Proteins
Singer & Nicolson (1972) Science 175:720-731
Proteins can be bound to membranes, integral
membrane protein, attached by a covalent lipid or
bound to another membrane protein
2011-12-08
Membrane Proteins
Singer & Nicolson (1972) Science 175:720-731
Reduction of Dimensionality
Non-covalent interaction of two macromolecules can be analyzed in terms of
a partition function of the following components:
Q = qct qt qr qv qc f qs
ct-intermolecular contacts
v-vibrations
t-translation
cf-conformational flexibility
(3)
r-rotation
s-solvation
Membranes 5325
Diffusion in the Membrane
Inclusions
Membrane Proteins
Membrane Proteins
Singer & Nicolson (1972) Science 175:720-731
Reduction of Dimensionality
Non-covalent interaction of two macromolecules can be analyzed in terms of
a partition function of the following components:
Q = qct qt qr qv qc f qs
ct-intermolecular contacts
v-vibrations
t-translation
cf-conformational flexibility
(3)
r-rotation
s-solvation
For comparison
Environment
Degrees of Freedom & Energy
Translational Rotational
Vibrational
Solution
3
3
6
8.4 kcal
8.2 kcal
-4.6 to -6.5 kcal
Membrane
2
1
3
5.6 kcal
2.7
-2.3 to -3.2 kcal
Binding Enhancement of 5 to 6.1 kcal
2011-12-08
Membrane Proteins
Membranes 5325
Diffusion in the Membrane
Inclusions
Membrane Proteins
Membrane Proteins
For comparison
Environment
Degrees of Freedom & Energy
Translational Rotational
Vibrational
3
3
6
8.4 kcal
8.2 kcal
-4.6 to -6.5 kcal
Membrane
2
1
3
5.6 kcal
2.7
-2.3 to -3.2 kcal
Binding Enhancement of 5 to 6.1 kcal
Solution
• Increased membrane viscosity
• Membrane crowding
• Cytoskeleton interactions
Cytochrome c picks electrons up at complex III and
delivers them to O2 at complex IV
2011-12-08
Membrane Proteins
Membranes 5325
Diffusion in the Membrane
Inclusions
Membrane Proteins
Membrane Proteins
• Increased membrane viscosity
• Membrane crowding
• Cytoskeleton interactions
Cytochrome c picks electrons up at complex III and
delivers them to O2 at complex IV
2011-12-08
Protein Diffusion
Membranes 5325
Diffusion in the Membrane
Inclusions
Protein Diffusion
Protein Diffusion
S-D Diffusion constant
D=
kB T
µh
log 0 − γ
4π µh
µa
P.G. Saffman and M. Delbrük (1975), Proc. Nat. Acad. Sci USA 72:3111-3113.
S-D Diffusion constant
kB T
µh
D=
log 0 − γ
4π µh
µa
P.G. Saffman and M. Delbrük (1975), Proc. Nat. Acad. Sci USA 72:3111-3113.
(3)
(3)
• Boundry conditions
• Lateral phase heterogenity
• Many membrane proteins are very immobile
• Some have a mobility that is S-D compatible
• Peripheral proteins are similar to soluble
proteins
2011-12-08
Protein Diffusion
Membranes 5325
Diffusion in the Membrane
Inclusions
Protein Diffusion
Protein Diffusion
• Boundry conditions
• Lateral phase heterogenity
• Many membrane proteins are very immobile
• Some have a mobility that is S-D compatible
• Peripheral proteins are similar to soluble
proteins
Alternative View
• Membranes are not a dilute solution
• Balance attraction and charge repulsion
• Lipids move with proteins
2011-12-08
Protein Diffusion
Membranes 5325
Diffusion in the Membrane
Inclusions
Protein Diffusion
Protein Diffusion
Alternative View
• Membranes are not a dilute solution
• Balance attraction and charge repulsion
• Lipids move with proteins
• Hydration and the assembly of membranes
Theoretically proposed as the basis for the
hydrophobic effect
Observed in the thermal behavior of the bilayers
2011-12-08
Interacting Bilayer membranes
Membranes 5325
Interaction
Interacting Bilayer membranes
Interacting Bilayer membranes
• Hydration and the assembly of membranes
Theoretically proposed as the basis for the
hydrophobic effect
Observed in the thermal behavior of the bilayers
• Hydration Energy and the size of bilayers
•
Osmotic Dehydration of monomer–polymer
distribution
2011-12-08
Interacting Bilayer membranes
Membranes 5325
Interaction
Interacting Bilayer membranes
• Hydration Energy and the size of bilayers
Interacting Bilayer membranes
•
Osmotic Dehydration of monomer–polymer
distribution
• Surface force measurment
Longer range forces (electrostatic, ion layers)
very strong short range forces
2011-12-08
Interacting Bilayer membranes
Membranes 5325
Interaction
Interacting Bilayer membranes
• Surface force measurment
Interacting Bilayer membranes
Longer range forces (electrostatic, ion layers)
very strong short range forces
• Pipette Aspiration
Evaluates the elasticity of the bilayer membrane
structure
2011-12-08
Interacting Bilayer membranes
Membranes 5325
Interaction
Interacting Bilayer membranes
• Pipette Aspiration
Interacting Bilayer membranes
Evaluates the elasticity of the bilayer membrane
structure
• Osmotic Dehydration
Application of solute that cannot enter the
bilayer spaces and therefore dehydrates the
bilayer structure
2011-12-08
Interacting Bilayer membranes
Membranes 5325
Interaction
Interacting Bilayer membranes
• Osmotic Dehydration
Interacting Bilayer membranes
Application of solute that cannot enter the
bilayer spaces and therefore dehydrates the
bilayer structure
• Dehydration promotes membrane fusion
2011-12-08
Interacting Bilayer membranes
Membranes 5325
Interaction
Interacting Bilayer membranes
Interacting Bilayer membranes
• Dehydration promotes membrane fusion
2011-12-08
Membrane Fusion
Membranes 5325
Interaction
Membrane Fusion
Schematic of membrane fusion
• Dehydration and
interaction
Membrane Fusion
• Role of membrane
fluctuations
• Curvature and lipid
structure
• Fusion”pore”
• Reversibility
Schematic of membrane fusion
• Dehydration and
•
•
•
•
interaction
Role of membrane
fluctuations
Curvature and lipid
structure
Fusion”pore”
Reversibility
Spontaneous curvature of membranes
Role of major lipid PE vs PC
Promote Inverted phase transition
2011-12-08
Membrane Fusion
Membranes 5325
Interaction
Membrane Fusion
Spontaneous curvature of membranes
Membrane Fusion
Role of major lipid PE vs PC
Promote Inverted phase transition
2011-12-08
Summary
Membranes 5325
Interaction
Summary
Summary