02 lipids dlevy
TRANSCRIPT
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Electron crystallographyUC DAVIS, 2006
Lipids in 2D crystallization
Daniel Lévy
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- Structure of lipids
- Properties of lipids
- Lipids in preparation of purified solubilized proteins
- Solubilisation of lipids by detergent
- Reconstitution of lipids upon detergent removal
- Lipids in 2D crystallization
- Lipid/detergent phases
- Lipid ligand for 2D crystallization
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2D crystallyzation of membrane proteins
Parameters
-Lipid/protein ratio-Type of lipids
-Type of detergent- Rate of detergent removal
- Buffer composition-T°C
- Inhibitors, substrates
detergent
lipid
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Amphiphiles
phosphatidyl choline lyso- phosphatidylcholine dodecylsulphate
Polar
Non-polar
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What’s the difference between amembrane lipid and a detergent?
• Water solubility.• Membrane lipids are soluble to about 10-9M• Detergents are soluble in the range 10-6 to 10-2M• This is because the non-polar regions of detergents are
smaller and only one alkyl chain• Lyso -phospholipids are detergents.
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LIPIDS
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Lα
Lc
Lβ’
θ
θ
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Transition Temperatures gel to liquid phase
Increases with the number of CH2
Increases with the unsaturation
Lipid extracts are in fluid phase
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Cell membrane compounds
---3070Chloroplast innermembrane
trace---
4522
5578
Mitochondrionouter membraneinner membrane
102664Golgi complex102762Endoplasmic reticulum23266Nuclear envelope
81043
43364279
49545418
Plasma membranes:red blood cellsliver cellsamoebamyelin
CarbohydrateLipidProteinMembranes
% of dried compoundsAdvanced Cell Biology ed. by L.M. Schwartz and M.M. Azar. Van Nostrand (New York; 1981).
Eucaryote
Lipid/protein < 0.3 - 4 w/w >
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Lipids are usually co-purified with solubilized proteinsand increase the protein stability
Wang lab,http://saturn.med.nyu.edu/research/sb/wanglab/
% monomer/polydispersity
Purification of Glut1 from human erythrocytes
Purification of the ABC transporter BmrA from B. subtillis expressed in E. coli (Ravaud, 2006)
Dimerization of PSII by DGDGKruse, 2000
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Lipids spontaneouly form bilayers in presence of water
The morphology of liposomes depends on methods of formation
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Cryo-EM is a powerfull technique for the characterization of the liposomes(Negatively stain usually leads to artefactual images of lipidsl)
unilamellar multilamellar
tubesAngular shaped
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OD
400
nmSolubilization of liposomes by detergent
For a lipid concentration of L (mM)Onset of solubilization at Dsat=Dw+Rsat (L)End of solubilization at Dsol=Dw+Rsol (L)
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DETERGENT DwatermM mg/ml
Rsatmol/mol w/w
Rsolmol/mol w/w
TRITON X100 0.18 0.12 0.64 0.5 2.5 2.0
C12E8 0.20 0.11 0.66 0.45 2.2 1.5
Octylglucoside 17 4.9 1.3 0.48 3.0 1.1
DDM 0.3 0.15 1 0.65 1.6 1.0
Cholate 3 1.29 0.3 0.16 0.9 0.5
The minimal amount of detergent needed to solubilized lipidsin 2D crystallization trials can be calculated (usefull for the dialysis)
For P=0.5mg/ml, LPR 0.5, Lipid 0.25 mg/mlFor full solubilization of lipid/protein inDDM 0.025%, OG 18mM
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- Fast equilibration of lipid/det/protein micelles (Bio-Beads): addition as liposomes- Taking care of the solubilization dynamic
- slow equilibration between micelles (dialysis)- Addition as solubilized lipids at Rsol
Kinetic of solubilization of lipid by detergents
OG
DDM
DOPC
DPPC
OG
C12E8 DDM
DOTM FOS-F16
AFM of planar lipid bilayer treated withdetergent at the cmc 4°C, 30 min
liposomes
micelles
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DIALYSIS
DILUTION
GEL CHROMATOGRAPHY
POLYSTYRENE BEADS
PROTEOLIPOSOMESMIXED MICELLES
Reconstitution by detergent removal
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Detergent removal by dialysis
Dialysis bagCut-off 14kD
High cmc detergents 1-2 daysLow cmc detergents 1-2 weeks
Simplicity and low costFlowthrough dialysis cellBio-Beads ouside
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COMPOUND ADSORPTIVE CAPACITY
(mg/g beads).
DETERGENT
TRITON X 100 185
C12 E8 190
DODECYL MALTOSIDE 105
CHOLATE 80
CHAPS, CHAPSO. 85
HECAMEG 110
OCTYL GLUCOSIDE 117
PHOSPHOLIPID
LIPOSOMES 1
LIPID-DETERGENT MICELLES
(Rsol)*
2
LIPID-DETERGENT MICELLE
(3. Rsol)
4
LIPID-DETERGENT-PROTEIN
MICELLES 0.5-1
PROTEIN
BR, Ca++ATPase, F0F1, melibiose
permease, cytochrome b6f
0-0.2
Detergent removal by Bio-Beads
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C12E8(0.2mM)
OG (17mM)Hecameg
DDM(0.2mM)
J.Struct.Biol (1997) 118, 226
Bio-Beads adsorb low andhigh cmc detergents
Time courses of detergent removal
Complete detergent removal using Bio-Beads and relative control of the ratedetergent removal
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detergent
lipid
Lipids in 2D trials
Preparation of lipids
Lipids are usually solubilized in CHCL3(in EtoH for cholesterol)-Keep at -80°C under Argon
For mixture of lipids, mix CHCL3-solubilized lipidsDry the solutionResuspend in water, vortex, sonicate with a tip sonicatorAliquot and freeze
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Statistically used lipids for crystallization
DMPC(C14, no insaturation)
DOPC (C18, insaturations)DOPC/DOPGDOPC/POPC
E.Coli lipids(Polar extract)
- not native membrane lipids- highly different lipids- no report with mixture containing cholesterol
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Membrane proteins are usually reconstituted in different kind of lipids at low LPR(in protein non-aggregation conditions)
Part I: lipids are not important for 2D crystallization
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Lipid/protein ratio is often the major parameter
LPR 0.35 w/wtubes
vesiclesLPR=0.25 w/w
PPase(thermotoga marinatus)
Stahlberg, 1998
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Proteins crystallized in different types of lipids
Even synthetic lipids lead to highest resolution 2D crystals
Gonen, 2005
Hasler, 1998
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D.Stokes, 1998
2D crystallisation of purified Ca-Atpasefrom sarcoplasmic reticulum induced by vanadate
Part II: Lipids are important for 2D crystallization
+ vanadate
- a) Specific defaults in lipid bilayer induce 2D crystallization
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Lipids as defaults in the bilayer
Lacapere, 1998
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T° phase transition(DMPC)
BR crystallisationinproteoliposomesof DMPC
25°C
4°C
Watts, A. 1995
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Micellar equilibration
Post-vesiculation
I
II
Bila
yer
clos
ure
III
Part III: lipid/detergent phases are crucial for 2D crystallization
Lipid/detergents intermediates are detergent and rate specifics
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Slow detergent removal at 4°C Fast detergent removal at 20°C
2D crystals of DDM purified Melibiose permease
The rate of detergent removal is a parameter of 2D crystallization
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Specific lipid/DDM or DOTM phases
Lambert, (1998)
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1µm
DOTM(BB)
DOM(BB)
OG/cholate(BB)
2D crystals of LH1-RC from Rb. sphaeroidesSame rate of detergent removal
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Octyl-thio-glucoside/lipid phase
Reconstitution of liposomes from different detergents solubilized lipids
OG DDM
LDAO LDAO + OTG
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250 nm
FhuA LH2
PSI
LDAO
LDAO+OTG
LDAO LDAO+OTG
DDM OTG
Chami, 2001
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Wilson-Kubalek, 2005
Lipid ligand mediated crystallizationPart I: helical crystallization
Hist-perfringolysin
Gal-cerebroside tubesDoped with Ni-NTA lipid
10µm
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Lipid ligand mediated crystallizationPart II: 2D crystallization of soluble proteins at theair/water interface
1
2
3Fromhertz, 1971, NatureUzgiris and Kornberg, 1983, Nature
Lipid ligands for specific recognition
- Negatively and positively charged- Lipid toxin receptor (GM1, Gb3)- substrate modified lipids
-Ni-NTA lipid for His-prot
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Binding of ternary micelles
Reconstitutionin lipid bilayer
2D crystallization
Detergent removal
Lipid ligand mediated crystallizationPart III: 2D crystallization of membraneproteins at the air/water interface
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Lipid/detergent interaction at the air/water interface
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Set-up for 2D crystallization by the lipid layer method
OM EM
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Characteristics of the lipid layer method - Protein concentration up to 20µgr/ml (1µg/trial)- Unic orientation of the proteins
Lipid layer In volume
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Hist-tag Membrane proteins crystallized using Ni-NTA DOGS
1µm1µm
BmrA
Nd (M.Chami)OmprN
25 Å(Senior)Pgp
Nd(S.Scheuring)
Aqp1
17 ÅBmrA
Nd (J.Walker)EF1FO
25 ÅTF1FO
15 ÅFhuA
ResolutionProteins
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Charged membrane proteins crystallized on oppositively charged lipid layer
BR
LH1-RCΔX
ANC2
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Conclusion
Characterization of the endogeneous lipids to improvethe stability of the purified proteins
Reconstitution are poorly specific to lipidsbut a large set of lipid increases the chance of 2D crystallization
Lipid/detergent intermediates are important andshould be study for any new detergent
Cholesterol and sphingomylin should be tried with eucaryot proteins