Biomedical Engineering Reference
In-Depth Information
namely distinguishing the seven sub-units of the oligomer can be achieved [99, 590].
The GroES ring acts like a 'lid' for the GroEL, sitting atop it under some conditions, and
the GroEL without the lid can be distinguished from the entire GroEL/GroES complex
[590], allowing the kinetics of the association/dissociation of the two partners to be
studied by high-speed AFM [304, 593], see Figure 7.18.
A major class of protein assemblies that have been extensively studied by AFM is that
of fibrillar assemblies, which have great importance for human health, due to their
implication in diseases such as Alzheimer's disease and their importance in blood clotting
[301]. For example, the formation of protein nets and fibrils formed during blood clotting
has been observed on mica and HOPG surfaces [594]. Collagen, the most abundant protein
in mammals, also forms fibrils. These have characteristic band structures, which are
simple to observe by AFM, and this has become a standard specimen for biological
AFM [35, 595-599].
Individual protein monomers can also be studied by AFM, although it is sometimes
difficult to image isolated molecules without adhering them to a surface [306]. One
preparation method commonly used for membrane proteins is to image them in a phos-
pholipid bilayer, which stabilizes them towards imaging, as well as mimicking biological
conditions. Proteins in lipid bilayers are covered in Section 7.3.3. Covalently binding
proteins to a surface can stabilize them toward single-molecule imaging, although this
might affect the protein structure. In some cases, careful control of pH and ionic strength is
enough to enable imaging of isolated proteins absorbed onto mica [301, 306].
III
I
II
IV
15 nm
5.s
5.s
I
III
5 nm
5 nm
10 nm
II
IV
Fig. 7.18. AFM imaging of single protein complexes. Left: high-resolution images (contact-mode
AFM) of GroEL (top) and GroEL/GroES assemblies (bottom). The association of the GroES onto the
GroEL adds about 5 nm to the height of the complex, and covers the internal cavity. Right: high-
speed imaging (IC-AFM) of GroEL/GroES association and dissociation. The images use a novel 1-D
imaging technique to increase speed. The slow scan axis was disabled, and the fast scan axis
(vertical) repeatedly scans a line containing various molecules. In the centre image (in the absence
of ATP), this leads to continuous stripes along the time axis (horizontal), as little association or
dissociation was occurring. When ATP is added (right image), the assembly heights switch rapidly
(see line scans below), indicating rapid association and dissociation. In both examples the images
were measured in buffer solutions. Adapted with permission from [590] and [304].
 
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