Chemistry Reference
In-Depth Information
13.6
Force as a Probe of Protein Chemistry
In the previous sections we have shown how force-clamp spectroscopy can be used to
probe the transition states of protein folding and unfolding reactions. An Arrhenius
term [63] offers a simple relationship between reaction kinetics and the physical
distance along a force-induced reaction coordinate to reach the transition state,
D
x. The
length-scale of this distance to the transition state was found to be between
8 Å [28] and 60 Å [104]) for protein folding, in rough agreement with the expected role of
long range hydrophobic forces [92]. For unfolding this distance was found to be much
shorter, in the range of
D
x
f
1.7 to 2.5 Å [50]. This distance is comparable to the size of
a water molecule, suggesting an important role for the solvent in the hydrogen bond
rupture necessary for unfolding [49]. However, it should be noted that protein
unfolding and refolding are complex processes, potentially involving hundreds to
thousands of atoms. Thus, it is dif
cult to precisely determine how an individual
interaction contributes to this transition state structure. In this section we show that
force-clamp spectroscopy can also be used to probe a simple system, composed of only
a few atoms, to carefully monitor the transition state structure of a chemical reaction.
We examine the reduction of individual disul
de bonds in a protein molecule,
identifying not only a transition state structure on a sub-Ångstrom scale, but also for
the
first time
finding how mechanical force can in
uence chemical kinetics [105].
A recent review by Beyer and Clausen-Schaumann covers the role of mechanical
forces in catalyzing chemical reactions [106]. The authors noted that a widespread
dif
culty in previous studies was that the reaction of interest could never be
consistently oriented with respect to the applied mechanical force and thus, the
in
uence of mechanical forces on these chemical reactions could not be studied
quantitatively [106]. Recently, we have shown that force-clamp spectroscopy can
overcome these barriers to directly measure the effect of a mechanical force on the
kinetics of a chemical reaction [105]. In these experiments, a disul
de bond was
engineered into a well-de
ned position within the structure of the I27 immunoglob-
ulin module of human cardiac titin (Figure 13.9A). Disul
D
x
u
de bonds are covalent
linkages formed between thiol groups of cysteine residues. These bonds are common
in many extracellular proteins and are important both for mechanical and thermo-
dynamic stability. The reduction of these bonds by other thiol-containing compounds
via an uncomplicated S
N
2-type mechanism [107
-
109] is common both in vivo and in
vitro; a commonly used agent is the dithiol reducing agent dithiothreitol (DTT). Using
force-clamp spectroscopy to extend single polyproteins of the modi
ed I27 we
rst
observe partial unfolding of individual modules up to the disul
de bond (
L1 in
Figure 13.9). The disul
de bond in I27 presents a covalent barrier to the complete
unfolding of the protein module. In the presence of DTT, this disul
de bond can be
reduced, allowing for extension of the residues trapped behind the disul
de bond
(
D
L
2
in Figure 13.9). Thus single disul
de reduction events are easily identi
ed by
observation of steps of length
D
L
2
in the unfolding trajectories. Tomeasure the rate of
reduction at a particular force and a particular concentration of reducing agent we
obtained an ensemble of single-molecule unfolding trajectories of the type seen in
D
