HIGH-SPEED ATOMIC FORCE MICROSCOPY FOR DYNAMIC BIOLOGICAL IMAGING - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 165

technological reasons described in the last section. Yet it would be valuable to
demonstrate the potential of high-speed AFM for observing dynamic events
of intermolecular interactions of proteins which take place in cell membrane
fractions.
The current view of cell membrane structure derives from the luid mosaic
model in which proteins are considered to diffuse freely within a luid lipid
bilayer. 13 The irst direct evidence for protein diffusion within cell membranes
was provided by hybrid cell experiments. 14 Since then, various techniques
including luorescence recovery after photobleaching microscopy 15 and
single particle tracking microscopy 16 have provided a more detailed
understanding of the mobile nature of proteins in biological membranes.
In particular, it has been shown that proteins in native membranes may not
diffuse freely but are in fact conined to speciic domains. Cells use several
conining mechanisms such as anchoring to the cytoskeleton through hetero-
bifunctional proteins, 17 diffusion barriers formed by the accumulation of
proteins anchored to cytoskeleton meshes 18 or self-assembly into large 2D
crystalline patches. Despite these advances, an understanding of membrane
dynamics at the nanoscale remains a major challenge primarily because of the
lack of measurement techniques allowing simultaneous spatial and temporal
observation of single molecules within native membranes.
In this section, we introduce the capability of high-speed AFM for
observing intermolecular interactions, lateral organization and rotational
dynamics in 2D protein crystals.

8.3.1 Defect Diffusion in Streptavidin 2D Crystals

For protein crystal formation, the protein-protein association energy must be
in an appropriate range. However, the association energy at each contact point
had not been assessed experimentally. Here, we show that high-speed AFM
imaging can enable its estimation using streptavidin as a model sample.

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Streptavidin is a protein that consists of four identical subunits: each
speciically binds to one biotin.

It is easily crystallized in a 2D form on
biotinylated lipid layers, which is considered to be an ideal model system to
investigate 2D crystals grown on lipid layers. On the biotinylated lipid layers,
two biotin-binding sites are occupied by the biotin moiety of a lipid layer,
while the other two are exposed to an aqueous environment and therefore
are free from biotin as depicted in Fig. 8.1a .
2D crystals of streptavidin were formed on a supported lipid bilayer (SLB)
as follows. The lipid composition used was dioleoylphosphatidylcholine
(DOPC), dioleoylphosphatidylserine (DOPS) and 1,2-dioleoyl-

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sn -glycero-

3-phosphoethanolamine- N -(cap biotinyl) (biotin-cap-DOPE) (7 : 2 : 1,