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