Lipids in 2D crystallization

Daniel Lvy

Electron crystallography UC DAVIS, 2006

- 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 -TC - Inhibitors, substrates

lipid detergent

Amphiphiles
Non-polar Polar

phosphatidyl choline

lyso- phosphatidylcholine

dodecylsulphate

Whats the difference between a membrane 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

Lc

Transition Temperatures gel to liquid phase

Increases with the unsaturation

Lipid extracts are in fluid phase

Increases with the number of CH2

Cell membrane compounds

Membranes Plasma membranes: red blood cells liver cells amoeba myelin Nuclear envelope Endoplasmic reticulum Golgi complex Mitochondrion outer membrane inner membrane Chloroplast inner membrane Protein 49 54 54 18 66 62 64 55 78 70 Lipid 43 36 42 79 32 27 26 45 22 30 Carbohydrate 8 10 4 3 2 10 10

Eucaryote

trace -----

% of dried compounds Advanced Cell Biology ed. by L.M. Schwartz and M.M. Azar. Van Nostrand (New York; 1981).

Lipid/protein < 0.3 - 4 w/w >

Lipids are usually co-purified with solubilized proteins and increase the protein stability
Purification of the ABC transporter BmrA from B. subtillis expressed in E. coli (Ravaud, 2006)

% monomer/polydispersity Purification of Glut1 from human erythrocytes Wang lab, http://saturn.med.nyu.edu/research/sb/wanglab/

Dimerization of PSII by DGDG Kruse, 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

Angular shaped

tubes

Solubilization 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)

OD 400nm

DETERGENT

Dwater mM mg/ml 0.18 0.20 0.12 0.11

Rsat
mol/mol w/w mol/mol

Rsol
w/w

TRITON X100

0.64 0.66

0.5 0.45

2.5 2.2

2.0 1.5

C12E8

Octylglucoside DDM Cholate

17 0.3 3

4.9 0.15 1.29

1.3 1 0.3

0.48 0.65 0.16

3.0 1.6 0.9

1.1 1.0 0.5

The minimal amount of detergent needed to solubilized lipids in 2D crystallization trials can be calculated (usefull for the dialysis) For P=0.5mg/ml, LPR 0.5, Lipid 0.25 mg/ml For full solubilization of lipid/protein in DDM 0.025%, OG 18mM

Kinetic of solubilization of lipid by detergents

- Fast equilibration of lipid/det/protein micelles (Bio-Beads) : addition as liposomes - Taking care of the solubilization dynamic
DOPC OG

liposomes

OG

DPPC C12E8 DDM

DDM
DOTM FOS-F16

micelles

- slow equilibration between micelles (dialysis) - Addition as solubilized lipids at Rsol AFM of planar lipid bilayer treated with detergent at the cmc 4C, 30 min

Reconstitution by detergent removal

DIALYSIS

GEL CHROMATOGRAPHY

DILUTION

POLYSTYRENE BEADS

MIXED MICELLES

PROTEOLIPOSOMES

Detergent removal by dialysis Dialysis bag Cut-off 14kD High cmc detergents 1-2 days Low cmc detergents 1-2 weeks Simplicity and low cost Flowthrough dialysis cell Bio-Beads ouside

Detergent removal by Bio-Beads

COMPOUND DETERGENT TRITON X 100 C12 E8 DODECYL MALTOSIDE CHOLATE CHAPS, CHAPSO. HECAMEG OCTYL GLUCOSIDE PHOSPHOLIPID LIPOSOMES LIPID-DETERGENT MICELLES (Rsol)* LIPID-DETERGENT MICELLE (3. Rsol) LIPID-DETERGENT-PROTEIN MICELLES PROTEIN BR, Ca++ATPase, F0F1, melibiose permease, cytochrome b6f

ADSORPTIVE CAPACITY (mg/g beads). 185 190 105 80 85 110 117

1 2 4 0.5-1 0-0.2

Bio-Beads adsorb low and high cmc detergents OG (17mM) Hecameg

C12E8 (0.2mM)

Complete detergent removal using BioBeads and relative control of the rate detergent removal

DDM (0.2mM)

Time courses of detergent removal

J.Struct.Biol (1997) 118, 226

Lipids in 2D trials

Preparation of lipids
Lipids are usually solubilized in CHCL3 (in EtoH for cholesterol) -Keep at -80C under Argon

lipid detergent

For mixture of lipids, mix CHCL3-solubilized lipids Dry the solution Resuspend in water, vortex, sonicate with a tip sonicator Aliquot and freeze

Statistically used lipids for crystallization DMPC

(C14, no insaturation)

DOPC (C18, insaturations) DOPC/DOPG DOPC/POPC

E.Coli lipids (Polar extract)

- not native membrane lipids - highly different lipids - no report with mixture containing cholesterol

Part I: lipids are not important for 2D crystallization

Membrane proteins are usually reconstituted in different kind of lipids at low LPR (in protein non-aggregation conditions)

Lipid/protein ratio is often the major parameter

LPR 0.35 w/w tubes PPase (thermotoga marinatus)

vesicles LPR=0.25 w/w

Stahlberg, 1998

Proteins crystallized in different types of lipids

Hasler, 1998

Even synthetic lipids lead to highest resolution 2D crystals

Gonen, 2005

Part II: Lipids are important for 2D crystallization

- a) Specific defaults in lipid bilayer induce 2D crystallization

+ vanadate
2D crystallisation of purified Ca-Atpase from sarcoplasmic reticulum induced by vanadate

D.Stokes, 1998

Lipids as defaults in the bilayer

Lacapere, 1998

T phase transition (DMPC)

25C

BR crystallisation in proteoliposomes of DMPC

4C

Watts, A. 1995

Part III: lipid/detergent phases are crucial for 2D crystallization Micellar equilibration I Bilayer closure

III

Post-vesiculation
Lipid/detergents intermediates are detergent and rate specifics

II

2D crystals of DDM purified Melibiose permease

Slow detergent removal at 4C

Fast detergent removal at 20C

The rate of detergent removal is a parameter of 2D crystallization

Specific lipid/DDM or DOTM phases

Lambert, (1998)

DOTM (BB)

DOM (BB)

OG/cholate (BB)

1m

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

Octyl-thio-glucoside/lipid phase

OG

DDM

LDAO

LDAO + OTG

Reconstitution of liposomes from different detergents solubilized lipids

FhuA
LDAO LDAO

LH2 LDAO+OTG

LDAO+OTG DDM OTG

250 nm

PSI

Chami, 2001

Lipid ligand mediated crystallization Part I: helical crystallization

Gal-cerebroside tubes Doped with Ni-NTA lipid

10m

Hist-perfringolysin

Wilson-Kubalek, 2005

Lipid ligand mediated crystallization Part II: 2D crystallization of soluble proteins at the air/water interface

Lipid ligands for specific recognition - Negatively and positively charged - Lipid toxin receptor (GM1, Gb3) - substrate modified lipids -Ni-NTA lipid for His-prot

3
Fromhertz, 1971, Nature Uzgiris and Kornberg, 1983, Nature

Lipid ligand mediated crystallization Part III: 2D crystallization of membrane proteins at the air/water interface

Binding of ternary micelles

Detergent removal

Reconstitution in lipid bilayer

2D crystallization

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 20gr/ml (1g/trial) - Unic orientation of the proteins

Lipid layer

In volume

Hist-tag Membrane proteins crystallized using Ni-NTA DOGS

Proteins FhuA TF1FO EF1FO Pgp Aqp1 BmrA OmprN

Resolution 15 25 Nd (J.Walker) 25 (Senior) Nd (S.Scheuring) 17 Nd (M.Chami)

1m
1m BmrA

Charged membrane proteins crystallized on oppositively charged lipid layer

BR

LH1-RC X

ANC2

Conclusion Characterization of the endogeneous lipids to improve the stability of the purified proteins Reconstitution are poorly specific to lipids but a large set of lipid increases the chance of 2D crystallization Lipid/detergent intermediates are important and should be study for any new detergent Cholesterol and sphingomylin should be tried with eucaryot proteins