Interaction of Melittin with Liposomes

Report
Morphological Changes of
Liposomes Induced by Melittin
Jirun Sun
Full Talk for Macromolecules
Seminar
J, Dufourcq et al., BBA 859, 1986, 33
Contents
• Background
– Introduction of Liposomes
– Introduction of Melittin
• Morphological changes of Liposomes with
Melittin
– Position of Melittin
– Pore Formation in Liposomes
– Morphological Changes of Liposomes
What Are Liposomes
• Artificial membrane vesicles.
• Drug delivery, chemical manufacturing ,
genetic engineering and so on
http://www.ncnr.nist.gov/programs/reflect/rp/biology/cell_membrane.html
Each phospholipid
includes
 a polar region:
glycerol, carbonyl of fatty
acids, Pi, & the polar head
group (X)
 2 non-polar hydrocarbon
tails of fatty acids (R1, R2).
Such an amphipathic lipid
may be represented as at
right.
O
O
R1
C
H2C
O
O
CH
H2C
C
R2
O
O
P
O
X
O
glycerophospholipid
3D picture of a
Phospholipid
O
O
R1
H2C
C
O
O
C
CH
H2C
R2
O
O
P
O
X
According to the head group
X, there are several kinds of
lipid.
O
OH
Me
X=
O
CH2
CH2
N
Me
O
NH3
O
CH
OH
Me
COO
Choline (PC)
Serine (PS)
OH
OH
OH
O
CH2
CH2
NH3
Ethanolamine (PE)
O
CH2
CH
CH2
OH
OH
Glycerol (PG)
Inositol (PI)
O-H
Acid(PA)
The membrane lipid
composition in an average
mammalian cell
O
Lipid
PC
PE
PI
PS
PA
cholesterol
%
45-55
15-25
10-15
5-10
1-2
10-20
O
R1
C
H2C
O
O
CH
H2C
C
R2
O
O
P
O
CH3
O
CH2
CH2
+
N CH3
CH3
phosphatidylcholine
Phosphatidylcholine (PC), or
lecithin, with choline as polar head
group.
It is a common membrane lipid.
Double bonds in fatty
acids usually have the cis
configuration.
Most naturally occurring
fatty acids have an even
number of carbon atoms.
Some fatty acids and their
common names:
14:0 myristic acid (DM);
16:0 palmitic acid (DP);
18:0 stearic acid (DS);
18:1 cisD9 oleic acid (DO)

4


3
2
O
C
1
O
fatty acid with a cis-D9
double bond
polar
"kink" due to
double bond
non-polar
DOPC: two oleic acid tails and a Choline head group
POPA: one P tail and one O tail, Acid head group
Liposomes Formed by Lipids
Bilayer
Micelle
Driven by hydrophilic and hydrophobic forces,
the nonpolar tails of lipids (U) tend to cluster
together, forming a lipid bilayer (1), a micelle (2).
The polar heads (P) face the aqueous
environment, Liposomes contain bilayers.
http://www.nupedia.com/newsystem/upload_file/830/bilayer_micelle.png
Size Determined by Methods
MLV: Multilamellar vesicles
SUV: Small unilamellar vesicles
LUV: Large unilamellar vesicles
GUV:Giant unilamellar vesicles
Sonication: SUV
Smaller than 100 nm diameter
Extrusion: LUV (Size depends on the filters)
100 nm—1 µm diameter
Evaporation: GUV
Larger than 1 µm diameter
http://www.avantilipids.com/PreparationOfLiposomes3Big.html
Preparation of Liposomes
Methods to Check the Morphology
of liposomes
• Freeze-fracture electron microscope
(FFEM) or electron microscope (EM)
• Light scattering (LS)
Freeze-fracture Electron
Microscopy
Freeze-fracture electron
Micrographs (a, b, c) and
negative staining (d) of
aqueous dispersions of
DOPC/DOPA (80:20 mol%)
after: 0(a); 10 (b and d) and
50 (c) cycles of freezethawing
Eur Biophys J 2000 29; 184
Static Light Scattering
Liposomes: DOPC
14 angles are checked
Top view of the geometry
around the sample cell
qi
Incident
beam
Test
tube
θ
qs
q
qi
Unscattered
beam
5.0
LnIntensity/Arbitrary
Indexmatching
liquid
4.8
Rg= 519±3 Å
4.6
4.4
4.2
4.0
3.8
0
100
300
2
Photodetector
ln(I ( q))  ln(I (0)) 
200
8
400
-2
q /10 cm
q2 Rg2
3
q
4n sin( / 2)
o
500
600
Dynamic Light Scattering (DLS)
Liposomes: DOPC
Six angles, 30,40,50,60,70
and 90 degree were checked.
1800
1600
Intensity Measured by Autocorrelation function
1400
-1
T
 I (t )  I (t   )d
Gamma/s
1
G(2)(t) = <I(0)I(t)> = lim
T  2T
T
The signal in the correlator is well
approximated by
1000
800
D=q
600
G(2)(t) = B(1 + fg(1)(t)2)
2
400
200
g(1)(t) is a simple exponential
0
0
g(1)(t) = e-t
100
200
2
2
Stokes’ law
Rh = 500±7 Å
1200
q kT
  q Dm 
6 R
2
8
300
-2
q /10 cm
400
What do SLS and DLS tell us?
Rh
Rg
If Rg/Rh ≈ 1 the
thickness of a ball is
about zero and then it
more like a thin bubble.
For liposomes, they are
Unilamellar
According to the DLS and
SLS data in the previous
two slides
Rg= 519±3 Å
Rh = 500±7 Å
Rg/Rh= 1.04
That DOPC is Unilamellar
Phase Transition Temperature (Tm)
• Important when preparing the
liposomes
• Important in the interactions of
Melittin with liposomes
liquid crystal
http://www.virtuallaboratory.net/Biofundamentals/lectureNotes/Topic2-3_Membranes.htm
crystal
Melittin
• It is the main component (50-60%) in bee
venom with 26 residues
• It has a very strong anti-inflammatory and
anti-bacterial effect
• It may cause allergic reaction, which can act
as a skin, eye or respiratory irritant.
• It is a cytolytic protein.
• It is an amphiphile protein.
http://molvis.chem.indiana.edu/C581_F97/protein_projects/Melittin.html
Tervilliger et.al., Nature, 1982, (299),371
The fact that melittin can
act as a lytic agent is
partly due to its detergentlike sequence.
Hydrophobic
Hydrophilic
∂ -helical wheel of the first 20 residues.
Leippe et al. PNAS, 88 (1991) 7659
Morphological Changes of
Liposomes with Melittin
• A weapon of bees
• The most popular model for studying
cytolytic activities
• Membrane fusion
• Drug releasing system
Well understanding of the interaction will be helpful on
•Building new instruments
•And studying new amphiphile proteins
Points
• Melittin position in liposomes
• The existence of poles
• Morphology changes of liposomes
– MLV or unilamellar
– Ratio of lipid to melittin
– Temperature
– Time
Melittin in Membranes
•Tetrameric aggregate
•End to end distance of
melittin from crystal
structure is about 3.6
nm
Simulation of melittin tetrameric aggregate
The N-terminus and the C-terminus are denoted
By N and C, respectively. The four helices are
Labeled 1-4.
Lin, et. al., Biophysical Journal, 78, 2000, 1714
Tervilliger et.al., Nature, 299, 1982, 371
Models of Pores
Toroidal model fits melittin better
Barrel-stave model
The difference is whether
the water core is lined by
both the peptides and the
lipid head groups
Toroidal model
Biophysical Journal, 81, 2001, 1475
Figures of Molecular
Dynamics Simulation
by Monte Carlo program
Snapshot of the pore from top (A)
and from side (B).
Lin, et. al., Biophysical Journal, 78, 2000, 1714
Existence of Poles
• Prepare liposomes in the presence of
fluorescent marker, like calcein
• Remove external calcein
• Measure background, set calcein
releasing as zero
• Add melittin
• Measure the released calcein by
spectrofluorimeter.
Leakage Experiments
Permeabilzing capacity
of melittin on MLV.
The release of calcein
from POPC/SMPC = 2:1
(mol/mol).
Melittin Concentration 1
µm.
G. Anderluh et. al., JBC, 278, 2003, 45216
Morphology Changes of Liposomes
• Temperature effects (Gel Phase or liquid crystal
phase)
• Unilamellar or multi-lamellar
• Ratio of lipid to melittin matters
• It is a dynamic process.
• Minor factors like buffer, pH values and so on
• That are combining results of all factors.
Melittin is Detergent-Like But They
Are Not the Same
Fusion happens!
Note for the cartoon:
DPPC LUV
L:M = 200
L:D = 200
Incubation: heat to
T>Tm and hold there for
a while
and then cool down
and measure at T<Tm.
No Fusion
C. G. Morgan et. al., BBA 732 (1983)668
Hydrodynamic Radius/ Å
Morphology Changes at Different
Ratios
Temp: 21 oC
Rh by checked by
Quasi-elastic LS
EPC: Egg PC
EPC is in LC state
at 21 oC
10000
EPC MLV
EPC SUV
L/M=25
L/M=13
1000
L/M=17
L/M=7
100
L/M=3
10
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Melittin/liposomes (Mol Ratio)
Data digitalized according to: J, Dufourcq et al., BBA 859, 1986, 33
Different L/M Ratios of
EPC SUV Seen by FFEM
A: Pure EPC SUV
B: L:M = 200
C: L:M = 30
D: L:M = 5
Magnification: 50,000x
From J, Dufourcq et al., BBA 859, 1986, 33
Different L/M Ratios of EPC MLV Seen
by FFEM
A: Pure EPC MLV
B: L:M = 30
C: L:M = 15
Magnification: 50,000x
From J, Dufourcq et al., BBA 859, 1986, 33
The Different Between MLV and
SUV
MLV + Melittin Vesicularisation
Fragmentation
LUV+ Melittin
SUV + Melittin
Fusion
J, Dufourcq et al., BBA 859, 1986, 33
Tiny particles
Gel Phase or LC Phase
LC phase: Morphology changes, Melittin
binds to vesicles
Gel phase: Only SUVs are morphologically
affected
Incubation: Morphology changes
It is not reversible!
Morphological Changes
at Different Temperatures
Scattered intensity
(Arbitrary)
12
Large particles scatter more!
10
Liposome used DPPC MLV,
Ratio of liposomes to melittin
L:M=20
Heating
Cooling
8
6
4
Tm for DPPC is 41 oC
2
0
0
10
20
30
40
50
o
Temp/ C
Data digitalized according to: J, Dufourcq et al., BBA 859, 1986, 33
Morphological Changes Detected
by Freeze-fracture EM
DPPC MLV
DPPC: melittin = 30
A: temp 20 oC
B: temp 50 oC
C: temp 20 oC after
incubation at 50 oC
From J, Dufourcq et al., BBA 859, 1986, 33
It Is A Dynamic Process
One of two
“Delta Dawns”
at LSU
1100
Procedure:
•Measure background
•Normalize with Tol or Emulsion
•Measure sample
•Data processing
Rg/cmX10
-8
1000
900
800
Size changing of liposome
(DOPC LUV) by adding
melittin checked by Rapid
SLS using a commercially
available, 18-detector
instrument
Melittin Added
No Stirring
L:M=70:1
Liposome
700
After Stirring
L:M=70:1
600
10
A touch is needed!
15
Time/ min
Data from Dr Russo’s Lab agree with the results in literature: J, Dufourcq et al., BBA 859, 1986, 33
20
Conclusion
– Liquid crystal state has more obvious
morphology changes than gel state does.
– The ratio of L/M matters
– MLV, SUV behave differently with melittin
– It is a dynamic process
Thanks

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