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to be acquired with subnanometer resolution and under physiological
conditions. Topographs of the extramembraneous domains of membrane
proteins can be acquired with a vertical resolution of 1 Å providing ine
details of the protruding protein domains above the membrane. Information
about the oligomeric state of a single protein, the organization of individual
components within multi-protein assemblies, as well as the individual
β -turns and loops connecting transmembrane
A
-helices can be obtained. 33
In the challenging context of the structural analysis of membrane proteins
only ~200 structures are currently available in the protein data bank),
AFM is a powerful tool to analyse proteins reconstituted into artiicial
membranes. Puriied and detergent-solubilized membrane proteins can be
reconstituted by detergent removal in the presence of additional lipids, at
high protein density, allowing the formation of 2D crystals in the plane of
the membranes.
These protein-enriched membranes can be prepared on
mica using appropriate buffers (generally, Tris supplemented with KCl and
MgCl 2 ) and imaged at high resolution with AFM. Some remarkable examples
include the characterization of the dimeric PufX-containing core complex of
Rhodobacter blasticus
50
the irst
view of the trimeric structure AmtB, an archetypal member of the ammonium
transporter family,
of the bacterial photosynthetic apparatus,
51
52
or the identiication and structure of a putative Ca
2+
-
binding domain at the C terminus of aquaporin 1. 53
Starting from pure lipid bilayers supported on mica, we have developed
in collaboration with Levy's group a new method for the reconstitution of
transmembrane proteins. It is based on previous studies of direct incorporation
of membrane protein into liposomes for functional studies. 54 The principle
relies on the direct incorporation of puriied membrane proteins in pre-formed
SLBs destabilized by sugar-based detergent. As a main result, the amount
of puriied proteins per trial is less than 1 picomole, far below the amount
requested in 2D or 3D crystallization, thereby allowing the AFM analysis of
eukaryotic membrane proteins that are dificult to overexpress.
Two large
membrane complexes, the light-harvesting 1 (LH1) and LH2 complexes from
the bacterial photosynthetic apparatus as well as the bacterial LacY permease,
have been successfully incorporated and imaged with a lateral resolution
in the nanometer range ( Fig. 1.4 ) . This resolution can be achieved because
lateral segregation and packing of incorporated transmembrane proteins
are possible within SLBs. This also supports the idea that, under appropriate
conditions, the lipid polar head-support interaction can be minimized and
that the water layer at the inner lealet-support interface is suficient for
allowing membrane diffusion.
10
 
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