Structural studies of lipid systems
Regine Willumeit
Hamburg, 22.10.2012
Introduction
What is important to know about lipids?
What can be measured?
Example for structure determination
1
Lipids are important for:
Cells -> membranes
As co-factors or as HDL (lipoproteins)
Joints
Crystallisation
in sensors
Polymeric implants (MPC polymers)
Biomembranes
2
Lipids in Cells
@Lehninger Biochemistry
Composition of an Erythrocyte Membrane
PE
PC
unknown
unknown
cell recognition
PS
apoptosis signal
intra celluar signals
@Lehninger Biochemistry
3
Lipid Names
Phospholipids = 4 letter code
first two letter: chains
last two letter: head group
POPC: Palmitoyl-Oleoyl Phosphatidyl Cholin
Structure of a Phospholipid
PE
PC
PS
4
Structure of a Phospholipid: Headgroups
neutral
neutral
Zeta Potential:
PC = 0
neutral
negative charge
PE = -25
PG = -60 mV
@Avanti Polar Lipids
Phosphatidylcholine (Symmetric Fatty Acid)
1,2-Diacyl-sn-Glycero-3-Phosphocholine
Biomembranes (Saturated Series)
Carbon
Number
3:0
4:0
5:0
6:0
7:0
8:0
9:0
10:0
11:0
12:0
13:0
14:0
15:0
16:0
Trivial
IUPAC
M.W.
Propionoyl
Butanoyl
Pentanoyl
Caproyl
Heptanoyl
Capryloyl
Nonanoyl
Capryl
Undecanoyl
Lauroyl
Tridecanoyl
Myristoyl
Pentadecanoyl
Palmitoyl
Trianoic
Tetranoic
Pentanoic
Hexanoic
Heptanoic
Octanoic
Nonanoic
Decanoic
Undecanoic
Dodecanoic
Tridecanoic
Tetradecanoic
Pentadecanoic
Hexadecanoic
369.35
397.41
425.46
453.51
481.57
509.62
537.67
565.73
593.78
621.84
649.89
677.94
706.00
734.05
16:0 [(CH3)4]
Phytanoyl
3,7,11,15-tetra
methylhexadecanoic
846.27
17:0
18:0
19:0
20:0
21:0
22:0
23:0
24:0
Heptadecanoyl
Stearoyl
Nonadecanoyl
Arachidoyl
Heniecosanoyl
Behenoyl
Trucisanoyl
Lignoceroyl
Heptadecanoic
Octadecanoic
Nonadecanoic
Eicosanoic
Heneicosanoic
Docosanoic
Trocosanoic
Tetracosanoic
762.10
790.16
818.21
846.27
874.32
902.37
930.43
958.48
DM
DP
5
@Avanti Polar Lipids
Phosphatidylcholine (Symmetric Fatty Acid)
1,2-Diacyl-sn-Glycero-3-Phosphocholine
(Unsaturated Series)
Biomembranes
Carbon Number
Trivial
IUPAC
M.W.
14:1
Myristoleoyl
9-cis-tetradecenoic
673.91
14:1
Myristelaidoyl
9-transtetradecenoic
673.91
16:1
Palmitoleoyl
9-cis-hexadecenoic
730.02
16:1
Palmitelaidoyl
9-transhexadecenoic
730.02
18:1
Petroselinoyl
6-cis-octadecenoic
786.13
18:1
Oleoyl
9-cis-octadecenoic
786.15
18:1
Elaidoyl
9-transoctadecenoic
786.13
18:2
Linoleoyl
9-cis-12-cisoctadecadienoic
782.09
18:3
Linolenoyl
9-cis-12-cis-15cisoctadecatrienoic
778.06
20:1
Eicosenoyl
11-cis-eicosenoic
842.23
20:4
Arachidonoyl
5,8,11,14(all -cis)
eicosatetraenoic
830.14
22:1
Erucoyl
13-cis-docosenoic
898.34
22:6
DHA
4,7,10,13,16,19 (all
-cis)
docosahexaenoic
878.18
24:1
Nervonoyl
15-cistetracosenoic
954.45
@Avanti Polar Lipids
Phosphatidylcholine (Asymmetric Fatty Acid)
Biomembranes
1-Acyl-2-Acyl-sn-Glycero-3-Phosphocholine
Carbon
Number
14:0-16:0
1-Acyl
2-Acyl
M.W.
Myristoyl
Palmitoyl
706.00
14:0-18:0
Myristoyl
Stearoyl
734.05
16:0-14:0
Palmitoyl
Myristoyl
706.00
16:0-18:0
Palmitoyl
Stearoyl
762.10
PM
PS
16:0-18:1
Palmitoyl
Oleoyl
760.09
PO
16:0-18:2
Palmitoyl
Linoleoyl
758.07
16:0-20:4
Palmitoyl
Arachidonoyl
782.09
16:0-22:6
Palmitoyl
Docosahexaenoyl
806.12
18:0-14:0
Stearoyl
Myristoyl
734.05
18:0-16:0
Stearoyl
Palmitoyl
762.10
18:0-18:1
Stearoyl
Oleoyl
788.14
18:0-18:2
Stearoyl
Linoleoyl
786.13
18:0-20:4
Stearoyl
Arachidonoyl
810.15
18:0-22:6
Stearoyl
Docosahexaenoyl
834.17
18:1-14:0
Oleoyl
Myristoyl
732.03
18:1-16:0
Oleoyl
Palmitoyl
760.09
18:1-18:0
Oleoyl
Stearoyl
788.14
SO
6
Biomembranes
@Lehninger Biochemistry
Amphipatic Molecules in Solution
hydrophilic
hydrophobic
CMC =
critical micelle concentration
highly diluted system
increase of concentration
@Lehninger Biochemistry
7
Amphipatic Molecules in Solution
POPC
POPG
inverse POPE
DPPE, DPPC, POPC, POPE….
@Lehninger Biochemistry
What can be measured?
monoolein/water
Martin Caffrey & Vadim Cherezov, Nat Protoc. 2009;4(5):706-31.
8
What can be measured?
Phase Behaviour of
Phospholipids
Data Base: LIPIDAT
PO=Palmitoyl-oleoyl (16:0-18:1); DP=Dipalmitoyl (16:0)
Lamellar Phase
Repeat Distance d
Bragg-equation:
n = 2d·sin
Q = 2 k0 sin =
4

sin
Seddon Handbook of Biol Physics 1995
9
Small Angle Scattering on
Lipid Vesicles also is Diffraction
Lipid
unilamellar
vesicle
multilamellar
vesicle
total ensemble
SAS
Diffraction
http://www.encapsula.com/products_01.html and Lehninger Biochemistry
1. Order
Diffraction of POPG Membranes (Neutrons)
Intensity
Biomembranes
q-value
10
1. Order
Diffraction of POPG Membranes (Neutrons)
Biomembranes
Intensity
Peaks equidistant
Repeat distance = 2 / q
Peak Distance = Repeat distance/n
n = 1, 2, 3, 4,….
Intensitiesq-value
& Phases ->
Scattering length density profile
11
Biomembranes
(invers) hexagonal phase
POPE Membrane (SAXS)
Biomembranes
(invers) hexagonal phase
POPE Membrane (SAXS)
Peaks NOT equidistant
Repeat distance = 2 / q
Peak distance = Repeat distance/n
n = 1, 3, 2, 7, 3, 12, 13, …
12
Cubic Phase
Seddon Handbook of Biol Physics 1995
Cubic Phase
Peaks NOT equidistant
Repeat distance = 2 / q
Peak distance = Repeat distance/n
n = 1, 2, 3, 2, 5, 6, 8, 3, …
Seddon Handbook of Biol Physics 1995
13
Sample Preparation
Solution of lipids in chloroform or methanol oder mixture
Drying of solution to obtain a lipid film
Hydratisation of the film with water / solvent
Liposomes
Drying of vesicles solution on support
Multilamellar Layers
Example for structure determination
Peptide Antibiotics
'Infectious diseases are the leading cause of world-wide and third
leading cause of death in the United States'
J.M. Hughes, Director of NCID & CDC, 1999
Main problem: fast acquisition of antibiotic resistance by bacteria
Possible alternative to 'classic' antibiotics: discovery of natural and
synthetic antibiotic peptides
Belong to the innate immune system in most species
14
Peptide Antibiotics
Biomembranes
Melittin
Magainins
(Xenopus laevis,
Bombina variegata)
Thionine
Plant Defensines
(Heuchera sanguinea)
Cecropins
(Hyalophora cecropia)
What is known about Peptide Antibiotics?
'Killing' mechanism:
Destruction of cytoplasmic membrane of bacteria
NO protein receptor!
No resistance??
Hypothesis:
physical interaction with the lipids of the membrane
15
The peptide NK-2
NK-lysin
Isolated from pig small intestine
78 aminoacids ca. 8.9 kDa
33% identity to a gene product (NKG5) from activated T and NK cell
helix 3+4 = NK-2 (res. 39-65)
Function: antibacterial, cytotoxic
5 -helices
hydrophilic
amphipathic
hydrophobic
10 positive charges
J. Andrä et al. Med Microbiol Immunol 188 (1999) 117-124
Common features
of peptide antibiotics:
The peptide NK-2
small (15-30 AA)
NK-lysin
highly amphipathic
Isolated from pig
small intestine ) charged
(positively
78 aminoacids ca. 8.9 kDa
NK-2:
good antibacterial activity (MIC < 1M)
hydrophilic
little hemolytic
amphipathic activity (>> 10M)
5 -helices
little cytotoxicity (>> 10M)
33% identity to a gene product (NKG5) from activated T and NK cell
helix 3+4 = NK-2 (res. 39-65)
Function: antibacterial, cytotoxic
Can we explain
Selectivity
and
hydrophobic
Mode of Action10
? positive charges
J. Andrä et al. Med Microbiol Immunol 188 (1999) 117-124
16
Membrane Composition
PG
Gram-negative
E.coli IM
S. typhimurium
P. cepacia
Gram-positive
S.aureus
B. subtilis
PE
CL
PC
SM PS
6
33
18
82
60
82
12
7
0
0
0
0
0
0
0
0
0
0
57
29
0
10
43
47
0
0
0
0
0
0
0
0
70
30
0
0
4
33
15
24
11
13
negative
C.albicans
Erythrocyte
PG = Phosphatidyl-glycerole
CL = Cardiolipin
SM = Sphingomyeline
PE = Phosphatidyl-ethanolamine
PC = Phosphatidyl-choline
PS = Phosphatidyl-serine
SAXS Results: POPC in 10 mM NaPhosphate-Buffer, pH 7.4
17
SAXS Results: DPPC in 10 mM NaPhosphate-Buffer, pH 5.2
pH 5.2
SAXS Results: POPG in Water
18
SAXS Results: POPG in Water
DMPG (negative) - FTIR
Stiffening
Decrease of 
=
Increase of ordering
L
L
DM=Dimyristoyl (14:0)
Willumeit et al. BBA 1669 (2005) 125 – 134
s(CH2) = symmetric stretching vibration
19
SAXS Results:
POPE in 10 mM Na-Phosphate-Buffer, pH 7.4
POPE + NK-2 (1000:1)
POPE
NK-2Influence
induces
a negative
of NK-2
on POPEmembrane
curvature -> breaking of the membrane!
20
New derivatives of NK-2
NK-2-[CS]
Several derivatives, small (15-30 AA), highly amphipathic
(positively) charged
K I L R G V S K K I M R T F L R R I S K D I L T G
K
K
NK-CS
K I L R G V S K K I M R T R L R R I S K D I L T G
K
K
NKCS-[FR]
K I L R G V S K K I M R T F L R R I S K K
NKCS-[20K]
K I L R G V S K K I M R T F L R R
NKCS-[17]
K I L R G V S K K I M R T F
NKCS-[14]
L R R I S K D I L T G
K
K
NKCS-[15-27]
New derivatives of NK-2
POPE
7.5
POPE+NKCS-[14] 1000:1
7.0
POPE+NKCS-[17] 1000:1
repeat distance [nm]
6.5
POPE+NKCS-[20K] 300:1
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
45
50
55
60
65
70
temperature [°C]
75
80
85
21
Shift of HII transition
Activity
New derivatives of NK-2
Shift for PE lipids (HII phase) is towards
higher temperatures!
Use of X-ray scattering to aid the design
and delivery of membrane-active drugs
G. Pabst • D. Zweytick • R. Prassl • K. Lohner, Eur Biophys J (2012) 41:915–929
How could anaesthetics work?
2 mol% ketamine = IC50 for a channel
composed of bent helices
22
Lipid Rafts
Areas in membranes with specific lipid and protein composition
Combination of glycosphingolipids and protein receptors
Usually more densely packed and "floating" in the membrane
Homepage of Jarek Majewski of the Lujan Neutron Scattering Center at Los Alamos
Regulating the Size and Stabilization of Lipid Raft-like
Domains and Using Calcium Ions as Their Probe
Or Szekely, Yaelle Schilt, Ariel Steiner, and Uri Raviv, Langmuir 2011, 27, 14767–14775
ratio 1:1:1
POPC
DOPC
DPPC
23
Self-Assembled Multicompartment Liquid Crystalline
Lipid Carriers for Protein, Peptide, and Nucleic Acid
Drug Delivery
A. ANGELOVA et al., ACCOUNTS OF CHEMICAL RESEARCH 44(2) (2011) 147–156
Molecules included in membrane
can lead to channel swelling
Decrease in curvature
Better storage capabilities
Self-Assembled Multicompartment Liquid Crystalline
Lipid Carriers for Protein, Peptide, and Nucleic Acid
Drug Delivery
A. ANGELOVA et al., ACCOUNTS OF CHEMICAL RESEARCH 44(2) (2011) 147–156
Change of structure
Release of drug
24
Crystallizing membrane proteins using lipidic
mesophases
Martin Caffrey & Vadim Cherezov, Nat Protoc. 2009;4(5):706-31.
Cholesterol
Protein
-> precipitant solution
-> shift of equilibrium away
from stability in the cubic
membrane.
-> phase separation
-> protein molecules diffuse
from the continuous
bilayered reservoir into
the lattice of the
advancing crystal face
Lipidic cubic phase technologies for
membrane protein structural studies
Vadim Cherezov, Current Opinion in Structural Biology 2011, 21:559–566
25
Stability of the lipid layers under shear
Lubrication of synovial joints is most efficient
Friction coefficient in the range of 0.001-0.01
Constant under changing conditions
Combination of complex structure of cartilage and
self-assembled structures formed by phospholipids
and biomacromolecules
Four main biological components of synovial fluid




albumin
proteoglycans such as lubricin and aggrecan
polyglycosaminoglycan like hyaluronan
surface active phospholipids (SAPLs)
M. Kreuzer, M. Reinhardt, J. Stahn, M. Golub, R. Willumeit, R. Dahint, R. Steitz
Stability of the lipid layers under shear
AMOR, PSI
M. Kreuzer, M. Reinhardt, J. Stahn, M. Golub, R. Willumeit, R. Dahint, R. Steitz
26
Thank you for your attention
Tresset PMC Biophysics 2009 2:3 doi:10.1186/1757-5036-2-3 / Technical Brief 2012 Volume 4
27