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to transmembrane proteins, these proteins can be easily incorporated
into SLBs. Two AFM papers using DOPC/SM/Chl supported bilayers have
shown that intestine or placental AP (respectively IAP and PLAP) are mainly
associated with the
phase.
45,46
However, a study using SLB made of the same
lipid mixture and analysed with a combined AFM-luorescence correlation
spectroscopy (FCS) setup showed that PLAP preferentially partitions in
the
lo
ld
phase and that only 25% of the proteins are associated with the
lo
phase.
47
The authors proposed that AFM is not suitable for imaging dynamic
individual molecules, such as AP inserted in a luid phase.
47
It is obvious that
the scan rate can be a critical parameter to image fast dynamic processes, but,
in this case, the diffusion coeficients in luid and
phases are theoretically
comparable (the classical difference of dynamics between these two phases
is a factor of 2)
21
and cannot explain the absence of IAP in the luid phase
observed by AFM. Another explanation that can be postulated to describe
the different behaviours of IAP and PLAP is the fact that fatty acid chains of
the IAP GPI-anchor are more saturated than that of the PLAP enzyme.
48
Our
recent data strongly suggest that IAP interacts with the most ordered lipid
species present in the gel phases of membranes exhibiting phase separation,
probably to prevent any hydrophobic mismatch.
48
Moreover, using a GPI-
anchored form of the angiotensin-converting enzyme, it was found that an
anchor with a chain length of C18 or longer induces the protein to mainly
partition in microdomains enriched in brain SM (the fatty acid chains are
mainly C18:0 and C24:1) in DOPC/SM/Chl SLBs. Such a partition was no
more observed using synthetic palmitoyl or stearoyl SM.
49
Taken together these results indicate that particular attention has to be
paid to the choice of lipids as well as fatty acid chain composition of GPI-
anchored proteins. These different examples also strengthen the potency of
AFM to investigate lipid and protein partitioning within SLB but also highlight
the dificulties encountered in interpreting and comparing results, even with
simple binary or ternary mixtures of lipids. To be accurately compared, model
membranes need to be identical in composition and certainly supported on
the same material, and, if necessary, proteins should be incorporated using
a similar technique. In addition, one has to keep in mind that membrane
components are highly dynamic, even in a gel phase, meaning that increasing
temporal resolution of AFM is clearly a key issue in this ield.
lo
1.5 STRUCTURAL ANALYSIS OF MEMBRANE PROTEINS
Besides its important contribution to the ield of lipid microdomains, AFM is
also the only microscopy technique that allows images of membrane proteins
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