New methods of reconstitution
Molecular Microscopy of Membranes
2D crystallization of membrane proteins at the
lipid layer
2D crystallization of membrane proteins at the lipid layer has been originally developped by our
Team in 1999.
2D crystallization by the lipid layer
The principle derived from both 2D
crystallization of soluble proteins at the
lipid layer and 2D crystallization of
membrane protiens in bulk. The
principle relies on the specific binding
of ternary micelles of
lipid/protein/detergent to a
functionalized ipid layer present at the
air/water interface. After binding to the
lipid surface, detergent is removed with
Bio-Bead or cyclodextrins leading to the
reconstitution of membrane protein in a
lipid bilayer. Protein/Protein interaction
can occur leading to the formation of
2D crystals. Surface can be tranfered to
an EM grid for analysis
Main interest of the method:1) the specific recognition of the surface, e.g. a His-protein to a NiNTA DOGS lipid layer, lead to decrease the protein working concentration to less than 10
microgr/ml. 2) Due to the specific binding, proteins have a single orientation within the lipid
layer while reconstitution is often symetrical when perfomed in bulk (Levy, D.J. Struct. Biol.,
1999). This allowed to crystallized proteins with bulky extramembraneous domain, like
ATPsynthase or ABC transporters, that can not be crystallized in bulk.
Main improvements: 1) we developped an optical set-up to in situ screen the binding and the
formation of the reconstituted lipid layer (Dezi, J. Struct. Biol. 2011). 2) We developped a method
to transfer the reconstituted lipid bilayer to a hydrophobic wafer for the analysis by Atomic Force
Microscopy (Seentier, B. J. Mol. Recong. 2011). 3) We obtained similar 2D crystals of BmrA, an
ABC transporter after specific binding ot fully hydrogenated Ni-NTA-DOGS lipid and to fully
fluorinated Ni-NTA lipid (Hussein, J Org Chem. 2009). 4) The functionalized lipid layer can also be
used for the purification of membrane proteins from solubilized extract and single particle
analysis (Dezi, BBA, 2011)
Proteins crystallized by the method bu us * or other groups:
FhuA, bacterial porin (Levy, D.J. Struct. Biol., 1999)*
Thermophilic FIFO (Levy, D.J. Struct. Biol., 1999)*
E. coli FIFO (Arechaga, Struct. Biol.,2007)
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1
New methods of reconstitution
Molecular Microscopy of Membranes
Pgp (Lee, JH, J. Biol. Chem., 2002; 2008)
BmrA (Orelle, Biochemistry, 2008, Dezi, J. Struct. Biol. 2011)*
ADT/ATP carrier (Lévy, D. J. S. Biol., 2001)*
Bacteriorhodopsin (Lévy, D. J. S. Biol., 2001)*
WZA outer membrane lipoprotein (nesper, J. J. Biol. Chem. 2003)
Ryanodine receptor (Yin CC, J. Struct. biol. 2005)
OprN (Chami, M. Pers. Com)
Aqp1 (S. Scheuring, Pers. Com)
LH1-RC PufX deleted (Busselez, Pers. Com)*
Reconstitution of membrane proteins in GUVs
Reconstitution of membrane proteins in GUVS
Giant unilamellar vesicles (GUVs) are
convenient biomimetic systems of the
same size as cells that are increasingly
used to quantitatively address
biophysical and biochemical processes
related to cell functions. We
developped a method to incorporate
transmembrane
proteins in GUVs, based on concepts
developed for detergent-mediated
reconstitution in large unilamellar
vesicles. Reconstitution is performed
either by direct incorporation from
proteins purified in detergent micelles
or by fusion of purified native vesicles
or proteoliposomes in preformed GUVs.
Lipid compositions of the membrane
and the ionic, protein, or DNA
compositions in the internal and
external volumes of GUVs can be
controlled. Using confocal microscopy
and functional assays,we showthat
proteins are unidirectionally
incorporated in the GUVs and keep
their functionality. We have
successfully tested our method with
three types of transmembrane proteins.
GUVs containing bacteriorhodopsin, a
photoactivable proton pump, can
generate large transmembrane pH and
potential gradients that are lightswitchable and stable for hours.
GUVswith FhuA, a bacterial porin, were
used to follow the DNA injection by T5
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2
New methods of reconstitution
Molecular Microscopy of Membranes
phage upon binding to its
transmembrane receptor. GUVs
incorporating BmrC/BmrD, a bacterial
heterodimeric ATP-binding cassette
efflux transporter, were used to
demonstrate the protein-dependent
translocation of drugs and their
interactions with encapsulated DNA.
Our method should thus apply to a wide
variety of membrane or peripheral
proteins for producing more complex
biomimetic GUVs.
Related Publication: Dezi, M. PNAS 2013.
Reconstitution of membrane proteins in
liposomes
The complexity of most biological membranes makes it difficult to study these membrane
proteins in situ. Therefore, purification from the native membrane and further reincorporation of
a purified membrane protein into an artificial membrane continue to be crucial steps in studying
the function and structure of these molecules. The necessity for reconstitution arises because
many membrane proteins express their full activity only when correctly oriented and inserted in
a lipid bilayer. In particular, reconstitution has played a central role in identifying and
characterizing the mechanisms of action of membrane proteins with a vectorial transport
function. More generally, through biochemical and biophysical approaches, it has led to
important information about lipid–protein and protein–protein interactions as well as topological
and topographical features of different classes of membrane proteins. The reconstitution of
membrane proteins to form two-dimensional crystals confined in a membrane has led to
important high-resolution structural information by electron crystallography.
Pioneer researches of J.L. Rigaud and colleagues on the mechanisms of proteins insertion into
liposomes during reconstitution by detergent removal led us to propose experimental guidelines
for the reconstitution of any type of membrane proteins in well-defined proteoliposomes.
Reviews on reconstitution:
1) Rigaud, J. L. & Levy, D. (2003). Reconstitution of membrane proteins into liposomes.
Methods Enzymol 372, 65-86.
2) Rigaud, J., Chami, M., Lambert, O., Levy, D. & Ranck, J. (2000). Use of detergents in twodimensional crystallization of membrane proteins. Biochim Biophys Acta 1508, 112-28.
3) Rigaud, J. L., Pitard, B. & Levy, D. (1995). Reconstitution of membrane proteins into
liposomes: application to energy-transducing membrane proteins. Biochim Biophys Acta
1231, 223-46.
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New methods of reconstitution
Molecular Microscopy of Membranes
Detergent removal with Bio-Beads
1) Lambert, O., Levy, D., Ranck, J. L., Leblanc, G. & Rigaud, J. L. (1998). A new “gel-like” phase
in dodecyl maltoside-lipid mixtures: implications in solubilization and reconstitution studies.
Biophys J 74, 918-30.
2) Rigaud, J. L., Mosser, G., Lacapere, J. J., Olofsson, A., Levy, D. & Ranck, J. L. (1997). BioBeads: an efficient strategy for two-dimensional crystallization of membrane proteins. J Struct
Biol 118, 226-35.
3) Levy, D., Gulik, A., Seigneuret, M. & Rigaud, J. L. (1990). Phospholipid vesicle solubilization
and reconstitution by detergents. Symmetrical analysis of the two processes using
octaethylene glycol mono-n-dodecyl ether. Biochemistry 29, 9480-8.
4) Levy, D., Bluzat, A., Seigneuret, M. & Rigaud, J. L. (1990). A systematic study of liposome
and proteoliposome reconstitution involving Bio-Bead-mediated Triton X-100 removal.
Biochim Biophys Acta 1025, 179-90.
Reconstitution of specific proteins
1) Levy, D., Bluzat, A., Seigneuret, M. & Rigaud, J. L. (1995). Alkali cation transport through
liposomes by the antimicrobial fusafungine and its constitutive enniatins. Biochem Pharmacol
50, 2105-7.
2) Levy, D., Gulik, A., Bluzat, A. & Rigaud, J. L. (1992). Reconstitution of the sarcoplasmic
reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during
reconstitution procedures involving the use of detergents. Biochim Biophys Acta 1107,
283-98.
3) Levy, D., Seigneuret, M., Bluzat, A. & Rigaud, J. L. (1990). Evidence for proton
countertransport by the sarcoplasmic reticulum Ca2(+)-ATPase during calcium transport in
reconstituted proteoliposomes with low ionic permeability. J Biol Chem 265, 19524-34.
Formation of 2D crystals by detergent removal
1) Milhiet, P. E., Gubellini, F., Berquand, A., Dosset, P., Rigaud, J. L., Le Grimellec, C. & Levy, D.
(2006). High-resolution AFM of membrane proteins directly incorporated at high density in
planar lipid bilayer. Biophys J 91, 3268-75.
2) Levy, D., Chami, M. & Rigaud, J. L. (2001). Two-dimensional crystallization of membrane
proteins: the lipid layer strategy. FEBS Lett 504, 187-93.
3) Chami, M., Pehau-Arnaudet, G., Lambert, O., Ranck, J. L., Levy, D. & Rigaud, J. L. (2001).
Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane
proteins. J Struct Biol 133, 64-74.
4) Levy, D., Mosser, G., Lambert, O., Moeck, G. S., Bald, D. & Rigaud, J. L. (1999). Twodimensional crystallization on lipid layer: A successful approach for membrane proteins. J
Struct Biol 127, 44-52.
5) Lambert, O., Moeck, G. S., Levy, D., Plancon, L., Letellier, L. & Rigaud, J. L. (1999). An 8-A
projected structure of FhuA, A “ligand-gated” channel of the Escherichia coli outer membrane.
J Struct Biol 126, 145-55.
6) Rigaud, J. L., Mosser, G., Lacapere, J. J., Olofsson, A., Levy, D. & Ranck, J. L. (1997). BioBeads: an efficient strategy for two-dimensional crystallization of membrane proteins. J Struct
Biol 118, 226-35.
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New methods of reconstitution
Molecular Microscopy of Membranes
Reconstitution of membrane proteins in planar
lipid bilayer
Incorporation of membrane proteins in planar lipid
bilayer
We developped a new method of incorporation
of transmembrane proteins in planar lipid
bilayer starting from 1 pmol of solubilized
proteins. The principle relies on the direct
incorporation of solubilized proteins into a
preformed planar lipid bilayer destabilized by
dodecyl-b-maltoside or dodecyl-bthiomaltoside, two detergents widely used in
membrane biochemistry. Successful
incorporations are reported at 20C and at 4C
with three bacterial photosynthetic
multisubunit membrane proteins. Height
measurements by atomic force microscopy
(AFM) of the extramembraneous domains
protruding from the bilayer demonstrate that
proteins are unidirectionally incorporated within
the lipid bilayer through their more
hydrophobic domains. Proteins are
incorporated at high density into the bilayer
and on incubation diffuse and segregate in
protein close-packing areas. The high protein
density allows high-resolution AFM topographs
to be recorded and protein subunits
organization delineated. This approach
provides an alternative experimental platform
to the classical methods of twodimensional
crystallization of membrane proteins for the
structural analysis by AFM. Furthermore, the
versatility and simplicity of the method are
important intrinsic properties for the conception
of biosensors and nanobiomaterials involving
membrane proteins.
Related Publications:Milhet, P.E. Biophysical J. 2006. Berquand, Ultramicroscopy, 2007. Levy and
Milhiet. Methods Mol Biol. 2013
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