02 Lipids DLevy

Electron crystallographyUC DAVIS, 2006

Lipids in 2D crystallization

Daniel Lévy

- 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

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

Amphiphiles

phosphatidyl choline lyso- phosphatidylcholine dodecylsulphate

Polar

Non-polar

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.

LIPIDS

Lα

Lc

Lβ’

θ

θ

Transition Temperatures gel to liquid phase

Increases with the number of CH2

Increases with the unsaturation

Lipid extracts are in fluid phase

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 >

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

Lipids spontaneouly form bilayers in presence of water

The morphology of liposomes depends on methods of formation

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

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)

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

- 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

DIALYSIS

DILUTION

GEL CHROMATOGRAPHY

POLYSTYRENE BEADS

PROTEOLIPOSOMESMIXED MICELLES

Reconstitution by detergent removal

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

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

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

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

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

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

Lipid/protein ratio is often the major parameter

LPR 0.35 w/wtubes

vesiclesLPR=0.25 w/w

PPase(thermotoga marinatus)

Stahlberg, 1998

Proteins crystallized in different types of lipids

Even synthetic lipids lead to highest resolution 2D crystals

Gonen, 2005

Hasler, 1998

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

Lipids as defaults in the bilayer

Lacapere, 1998

T° phase transition(DMPC)

BR crystallisationinproteoliposomesof DMPC

25°C

4°C

Watts, A. 1995

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

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

Specific lipid/DDM or DOTM phases

Lambert, (1998)

1µm

DOTM(BB)

DOM(BB)

OG/cholate(BB)

2D crystals of LH1-RC from Rb. sphaeroidesSame rate of detergent removal

Octyl-thio-glucoside/lipid phase

Reconstitution of liposomes from different detergents solubilized lipids

OG DDM

LDAO LDAO + OTG

250 nm

FhuA LH2

PSI

LDAO

LDAO+OTG

LDAO LDAO+OTG

DDM OTG

Chami, 2001

Wilson-Kubalek, 2005

Lipid ligand mediated crystallizationPart I: helical crystallization

Hist-perfringolysin

Gal-cerebroside tubesDoped with Ni-NTA lipid

10µm

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

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

Lipid/detergent interaction at the air/water interface

Set-up for 2D crystallization by the lipid layer method

OM EM

Characteristics of the lipid layer method - Protein concentration up to 20µgr/ml (1µg/trial)- Unic orientation of the proteins

Lipid layer In volume

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

Charged membrane proteins crystallized on oppositively charged lipid layer

BR

LH1-RCΔX

ANC2

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