Chemistry Reference
In-Depth Information
unbinding force. The distribution of unbinding forces measured at various loading
rates, reffered as the dynamic force spectrum, yields the energy landscape for the
interaction (Figure 7.4D).
Contrary to the techniques described above, force spectroscopy gives information
on non-equilibrium reaction kinetics. A force F applied to a complex increases the
dissociation rate constant as represented below [131]:
;
x
b
F
k
B
T
k
diss
ð
F
Þ¼
k
diss
ð
0
Þ
exp
ð
7
:
10
Þ
where k
diss
(0) denotes the dissociation rate constant in the absence of an external
force, x
b
is the distance between the bound state and the transition state along the
reaction coordinate (direction of the applied force), k
B
is the Boltzmann constant, and
T is the absolute temperature. The product x
b
F corresponds to the decrease in the
height of the energy barrier when a mechanical force is applied. Values of k
diss
(0) for
different ligand
-
receptor complexes are shown in Table 7.2. Importantly, k
diss
(0) is
similar to the thermal (equilibrium) dissociation rate constant under the unique
conditions that ligand
-
receptor interactions are characterized by a one-barrier energy
landscape. Examples of ligand
-
receptor dissociation processes with multiple energy
barrier pro
les are also presented in Table 7.2 and in Figure 7.4 D.
The association kinetics can be characterized using single-molecule force mea-
surements by the adhesion probability P, i.e. the fraction of the force curves which
show a speci
c binding event. The association rate constant k
ass
can be determined by
integrating the differential equation with the appropriate boundary conditions:
dP
dt
¼
k
diss
P
þ
k
ass
c
eff
ð
1
P
Þ;
ð
7
:
11
Þ
where c
eff
denotes the effective concentration of binding partners in the accessible
volume between the AFM tip and the cell surface [132].
Interestingly, single-molecule force spectroscopy has essentially been applied to
the study of adhesion receptors. To our knowledge, the interaction between a GPCR
and its ligand has not been investigated in living cells. The mechanical unfolding of
single membrane proteins has recently been investigated by combining AFM
imaging and force spectroscopy [120]. Because the spectra showing unfolding events
contain information on the strength and location ofmolecular interactions within the
protein [133], the binding between a GPCR and its ligand could be studied using this
technique.
7.6.2
Novel AFM-based Techniques
Recently, we emphasized the potential of planar cell membranes deposited on
submicrometer-sized apertures in the study of molecular interactions with both
sides of integral membrane proteins [134]. In a similar approach, a promising two-
chamber AFM set-up has been designed, which allowed combined
uorescence
microscopy and high spatial resolution imaging of proteins spanning suspended
