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proteins. Spontaneous insertion of ion-channel proteins has been successfully
used, but this strategy cannot be applied to the majority of transmembrane
proteins. One of the promising ways is to form membrane by proteoliposomes
fusion. However, this approach presents important bottlenecks, such as the
puriication of proteins in large amounts. In addition, proteins generally
display two different membrane orientations, except in some detergent-
mediated reconstitution approaches. 54 Direct incorporation of membrane
proteins, similar to what we have developed for structural analysis purposes,
can be useful.
1.7 AFM METHODOLOGICAL DEVELOPMENTS
As we have seen, AFM represents a very powerful tool to explore the structure
of SLBs, whether or not containing proteins. However, commercial setups
cannot investigate membrane dynamics (lipids diffuse in the membrane
with a diffusion coeficient in the μm 2 /s range) because of the limit of the
tip scanning rate (typically between 0.5 and 7 Hz for this type of sample).
Recent advances have been made owing to the combination of AFM with FCS,
irst described in 2005. 63 Structural information and spatial distribution of
membrane components, i.e. microdomains, is given by AFM, whereas FCS
provides their local dynamic properties. This combination has been applied
to studying partitioning of GM1, Cer and AP into SLBs.
Nevertheless, the
FCS lateral resolution is still rather poor compared with that of AFM (the
detection area or beam waste diameter used in FCS experiments is larger than
200 nm). A very promising way to get simultaneous dynamics and topography
of SLBs is to break the speed limit of AFM. Important progress has been made
in cantilevers, scanners and controllers, 64 and two main setups allowing
video-rate imaging in liquid seem to be promising in the membrane ield. The
irst setup is a high-speed contact mode AFM developed by the Miles group
in Bristol 65 in which the sample is placed on a lexure stage that is aligned
with the fast-scanning
39,63
direction, and the cantilever is positioned on a piezo
tube that is used for slow-scanning
x
directions. This setup allows
video-rate acquisition of biological samples, but no images of membranes
have been reported so far. The second setup is a high-speed tapping mode
AFM developed by the group of Ando in Kanazawa 66 (see Chapter 8 ). In
order to increase the scanning rate in tapping mode, small cantilevers with
a high resonance frequency and a low spring constant (150-280 pN/nm and
1.3-1.8 MHz), as well as new AC-to-DC converters, scanners and dynamic
PID controllers, have been developed. Different biological systems such as
GroES/GroES or myosin motors have been investigated, providing real-time
y
and
z
 
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