Biomedical Engineering Reference
In-Depth Information
Fig. 7.27. Protein unfolding by AFM. Left: schematic of typical experiment, showing polyprotein
being stretched between AFM tip and a surface to which it is covalently bound. Top right: typical
force curve measured on a polyprotein in constant velocity mode. Bottom right: typical result from
the same sample in constant-force mode. Adapted with permission from [586].
function, and thus mechanical resistance is an important part of their design [719]. While
there are other techniques which may be used to study mechanical unfolding of proteins
(such as optical tweezers), the accessibility and simplicity of protein unfolding by AFM
has meant it is the most popular technique with which to study the phenomenon today
[717]. The folded structures of proteins are typically held together by forces such as
hydrogen bonding, hydrophobic, ionic, and van de Waals interactions, which are all weak
interactions, but are collectively strong enough to hold the structure together. Therefore,
the fine details of unfolding pathways require high force resolution. The way in which
traditional force spectroscopy works is not ideal for protein unfolding studies, because
normal force spectroscopy is carried out at a constant velocity. This means that in protein
unfolding, large changes in applied force will occur during the process. A method that was
developed to overcome this limitation is force clamp spectroscopy, in which an additional
force feedback loop is added to the instrument, to maintain a constant force during
unfolding [720, 721]. A further derivative of this technique is force ramp spectroscopy,
where the feedback loop is used to maintain a constant increase in force during unfolding.
At the time of writing, nearly all force-clamp spectroscopy has been carried out with
modified instruments [722], but commercial instruments with such capabilities have also
begun to appear. Typically such experiments are carried out with synthesized polypro-
teins, large molecules with multiple copies of a single protein domain [716, 718]. Pulling
this type of molecules should give rise to a characteristic 'fingerprint' force curve (i.e. the
sawtooth- or staircase-shaped profiles shown in Figure 7.27), which helps to reduce the
ambiguities in force spectroscopy data discussed above. A new generation of commercial
instruments have been produced recently, which are designed to optimize the ultimate
force resolution by reducing noise in the
z
axis, largely spurred by the requirements of
protein unfolding experiments. It has been estimated that using standard commercial
cantilevers, thermal noise limits the resolution to approximately 6 pN [723]. These
