Chemistry Reference
In-Depth Information
experimental tool which can probe protein folding at the single-molecule level and
has the ability to test the physical models proposed. For the
first time, unique insight
into the folding trajectory of a single protein can be gained using force-clamp
spectroscopy, marking the beginning of rigorous tests of the microscopic physical
mechanisms involved in protein folding. In the next section we will introduce the
force quench experiment, followed by a discussion of one particular physical model
for protein folding.
13.5.1
The Force Quench Experiment
The force quench experiment monitors the end-to-end length of a single poly-
protein during reversible unfolding
-
folding cycles [28]. In the example shown
(Figure 13.6A) the polyprotein is composed of nine repeats of the small protein
ubiquitin, held at a constant force of 110 pN for a
fixed time of 4 s. This applied force
results in the probabilistic unfolding of individual ubiquitinmodules, as described in
Section 13.2.2. After 4 s we reduce or quench the force from 110 to 15 pN. The
Figure 13.6 Force quench experiment reveals
the folding trajectory of a single polyprotein. (A)
The folding pathway of ubiquitin is directly
measured by force-clamp spectroscopy. The end-
to-end length of a protein is shown as a function
of time. The folding trajectory can be divided into
four distinct regions. The first stage (1) is due to
the elastic recoil of the unfolded polyprotein as
the force is reduced. The second stage (2) in the
folding trajectory begins at the end of the rapid
elastic recoil and is marked by a noticeable
increase in length fluctuations. This stage
relaxes with a slow rate of collapse. The
beginning of the third stage (3) is observed by an
abrupt change in the slope of the trajectory,
where the molecule contracts to a conformation
of an end-to-end length comparable to the
native state. Stages (2) and (3) vary greatly
between trajectories and cannot always be
distinguished. The final stage (4) is where the
protein acquires its mechanical stability through
the formation of the native contacts [28]. (B) The
corresponding applied force is also shown as a
function of time.
