Morphological Changes of Liposomes Induced by Melittin Jirun Sun Full Talk for Macromolecules...
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Transcript of Morphological Changes of Liposomes Induced by Melittin Jirun Sun Full Talk for Macromolecules...
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
O P O
O
O
H2C
CH
H2C
OCR1
O O C
O
R2
X
glycerophospholipid
Each phospholipidincludes 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 lipidmay be represented as atright. 3D picture of a
Phospholipid
O P O
O
O
H2C
CH
H2C
OCR1
O O C
O
R2
X
According to the head group X, there are several kinds of lipid.
X= O CH2 CH2 N
Me
Me
Me
O CH2 CH2 NH3O CH2 CH CH2
OH OH
O CH
NH3
COO
O
OH
OH
OH
OH
OH
Choline (PC)
Ethanolamine (PE) Glycerol (PG)
Serine (PS)
Inositol (PI)
O-HAcid(PA)
The membrane lipid composition in an average
mammalian cell
Lipid %
PC 45-55
PE 15-25
PI 10-15
PS 5-10
PA 1-2
cholesterol 10-20
O P O
O
O
H 2 C
C H
H 2 C
OCR 1
O O C
O
R 2
C H 2 C H 2 N C H 3
C H 3
C H 3
+
p h o sp h a tid ylc h o lin e
Phosphatidylcholine (PC), or lecithin, with choline as polar head group.
It is a common membrane lipid.
Some fatty acids and their common names:14:0 myristic acid (DM); 16:0 palmitic acid (DP); 18:0 stearic acid (DS); 18:1 cis9 oleic acid (DO)
Double bonds in fatty acids usually have the cis configuration.
Most naturally occurring fatty acids have an even number of carbon atoms.
C
O
O 1
23
4
fatty acid with a cis-9 double bond
DOPC: two oleic acid tails and a Choline head groupPOPA: one P tail and one O tail, Acid head group
Liposomes Formed by Lipids
http://www.nupedia.com/newsystem/upload_file/830/bilayer_micelle.png
Bilayer
MicelleDriven 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.
Size Determined by Methods
Sonication: SUV Smaller than 100 nm diameterExtrusion: LUV (Size depends on the filters) 100 nm—1 µm diameterEvaporation: GUV Larger than 1 µm diameter
MLV: Multilamellar vesiclesSUV: Small unilamellar vesiclesLUV: Large unilamellar vesiclesGUV:Giant unilamellar vesicles
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 freeze-thawing
Eur Biophys J 2000 29; 184
Static Light ScatteringTop view of the geometry around the sample cell
o
nq
)2/sin(4
3
))0(ln())(ln(22gRqIqI
q
θ
Photodetector
qs
Incident beam
Test tube
qi
qi
Index-matching liquid
Unscattered beam
0 100 200 300 400 500 6003.8
4.0
4.2
4.4
4.6
4.8
5.0
q2/108cm-2
LnIn
tens
ity/A
rbitr
ary
Liposomes: DOPC14 angles are checked
Rg= 519±3 Å
Dynamic Light Scattering (DLS)
hom R
kTqDq
6
22 Stokes’ law
Liposomes: DOPC Six angles, 30,40,50,60,70 and 90 degree were checked.
0 100 200 300 4000
200
400
600
800
1000
1200
1400
1600
1800
D=q2Gam
ma/
s-1
q2/108cm-2
Rh = 500±7 Å
G(2)(t) = <I(0)I(t)> = ) () (2
1
T
TT
dtItIT
lim
Intensity Measured by Autocorrelation function
G(2)(t) = B(1 + fg(1)(t)2)
The signal in the correlator is well approximated by
g(1)(t) is a simple exponential
g(1)(t) = e-t
What do SLS and DLS tell us?
Rg
Rh
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 slidesRg= 519±3 Å Rh = 500±7 ÅRg/Rh= 1.04That DOPC is Unilamellar
Phase Transition Temperature (Tm)
• Important when preparing the liposomes
• Important in the interactions of Melittin with liposomes
liquid crystal crystal
http://www.virtuallaboratory.net/Biofundamentals/lectureNotes/Topic2-3_Membranes.htm
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.htmlTervilliger et.al., Nature, 1982, (299),371
The fact that melittin can act as a lytic agent is partly due to its detergent-like sequence.
Leippe et al. PNAS, 88 (1991) 7659
∂ -helical wheel of the first 20 residues.
HydrophilicHydrophobic
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
Simulation of melittin tetrameric aggregateThe N-terminus and the C-terminus are denotedBy N and C, respectively. The four helices are Labeled 1-4.
Lin, et. al., Biophysical Journal, 78, 2000, 1714Tervilliger et.al., Nature, 299, 1982, 371
•Tetrameric aggregate•End to end distance of melittin from crystal structure is about 3.6 nm
Models of Pores
Barrel-stave model
Toroidal modelBiophysical Journal, 81, 2001, 1475
Toroidal model fits melittin better
The difference is whether the water core is lined by both the peptides and the lipid head groups
Snapshot of the pore from top (A) and from side (B).
Figures of Molecular Dynamics Simulation by Monte Carlo program
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
G. Anderluh et. al., JBC, 278, 2003, 45216
Permeabilzing capacity of melittin on MLV. The release of calcein from POPC/SMPC = 2:1 (mol/mol). Melittin Concentration 1 µm.
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.
C. G. Morgan et. al., BBA 732 (1983)668
Fusion happens!Note for the cartoon:DPPC LUVL:M = 200L:D = 200
No Fusion
Melittin is Detergent-Like But They Are Not the Same
Incubation: heat to T>Tm and hold there for a while and then cool down and measure at T<Tm.
Morphology Changes at Different Ratios
0.00 0.05 0.10 0.15 0.20 0.25 0.3010
100
1000
10000
L/M=3
L/M=17
L/M=7
L/M=13L/M=25
Hyd
rod
ynam
ic R
adiu
s/ Å
Melittin/liposomes (Mol Ratio)
EPC MLV EPC SUV
Temp: 21 oCRh by checked by Quasi-elastic LSEPC: Egg PC EPC is in LC state at 21 oC
Data digitalized according to: J, Dufourcq et al., BBA 859, 1986, 33
A: Pure EPC SUVB: L:M = 200C: L:M = 30D: L:M = 5Magnification: 50,000x
Different L/M Ratios of EPC SUV Seen by FFEM
From J, Dufourcq et al., BBA 859, 1986, 33
Different L/M Ratios of EPC MLV Seen by FFEM
A: Pure EPC MLVB: L:M = 30C: L:M = 15Magnification: 50,000x
From J, Dufourcq et al., BBA 859, 1986, 33
The Different Between MLV and SUV
MLV + Melittin
LUV+ Melittin Tiny particles
Fragmentation
SUV + MelittinFusion
Vesicularisation
J, Dufourcq et al., BBA 859, 1986, 33
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
Data digitalized according to: J, Dufourcq et al., BBA 859, 1986, 33
Liposome used DPPC MLV,Ratio of liposomes to melittinL:M=20
0 10 20 30 40 500
2
4
6
8
10
12
Sca
ttere
d in
tens
ity
(Arb
itrar
y)
Temp/oC
Heating Cooling
Tm for DPPC is 41 oC
Large particles scatter more!
Morphological Changes Detected by Freeze-fracture EM
DPPC MLVDPPC: melittin = 30A: temp 20 oCB: temp 50 oCC: 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
Size changing of liposome (DOPC LUV) by adding
melittin checked by Rapid SLS using a commercially
available, 18-detector instrument
10 15 20
600
700
800
900
1000
1100
After StirringL:M=70:1
Melittin AddedNo StirringL:M=70:1
LiposomeR
g/cm
X10
-8
Time/ minA touch is needed!
Procedure: •Measure background•Normalize with Tol or Emulsion •Measure sample •Data processing
Data from Dr Russo’s Lab agree with the results in literature: J, Dufourcq et al., BBA 859, 1986, 33
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