Chemistry Reference
In-Depth Information
through a linker in a desired orientation. Amembrane surface with zwitterionic polar
head groups such as phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE)
is known to resist protein adsorption (e.g. [38, 39]). Streptavidin is also useful for
speci
c attachment of biotinylated protein because it also resists non-speci
c protein
adsorption. Mica surface-supported planar lipid bilayers [40] can be easily formed
from liposomes because their strong hydrophilic interaction disrupts the lipo-
somes [41]. Various lipids with functional groups that are attached to polar groups
(e.g. biotin attached to PE; Ni-NTA attached to PS) are available and can be used to
accomplish the speci
c attachment of proteins labeled with biotin or his-tags to
planar lipid bilayers. DOPC lipids are useful for preparing streptavidin 2D crystals
when used together with biotinylated lipids [42, 43]. These lipids contain an
unsaturated hydrocarbon on each of the two alkyl chains, which causes bending
of the chains, and therefore weakens the interaction between neighboring DOPCs.
This weak interaction lowers its phase-transition temperature to
20
C, which
affords a large
fluidity to the planar bilayer at room temperature and thus facilitates
2D-crystal formation of streptavidin (Figure 12.7a). The densely packed streptavidin
does not diffuse easily. If less diffusion is necessary, the packed streptavidin can
be crosslinked with glutaraldehyde, which does not affect its ability to bind to biotin.
We used this surface for the selective attachment of biotinylated GroEL in a side-on
orientation. DPPC contains no unsaturated hydrocarbons in the alkyl chains,
therefore its phase-transition temperature is high (
41
C) making DPPC appropri-
ate for preparing planar bilayers with a low
uidity. For example, when planar bilayers
are formedwithDPPCat a high temperature (
60
C) together with a certain fraction
of DPPE
-
biotin, streptavidin that is sparsely attached to the surface barely diffuses at
room temperature (Figure 12.7b). When DOPE
-
biotin is used together with DPPC,
the sparsely attached streptavidin diffuses at a moderate speed (Figure 12.7c).
12.9
Future Prospects
High-speed AFM imaging studies are a nascent
field. Therefore, the number of
researchers and imaging data that have been successfully captured on video are quite
limited. However, it seems very certain that this new microscopy will be used widely
in the biological sciences. High-speed AFM will become commercially available
probably in 3
-
5 years. Presently, though, the force exerted between the oscillating tip
and sample is barely able to preserve weak protein
-
protein associations. Moreover,
the imaging rate is not high enough to study fast biological processes. For high-speed
AFM to become truly useful for a wide variety of biological systems, reduction in the
force and increase in the imaging rate are essential.
How much better can the imaging rate and tip-sample interaction force be? The
cantilever is the factor limiting both requirements. Generally, to improve the
resonant frequency the rigidity must be compromised and vice versa. High-speed
nc-AFMwill allow the use of more rigid cantilevers with higher resonant frequencies
without concerns about disrupting delicate samples. It also has the potential to image
