Chemistry Reference
In-Depth Information
coordinate, but are nevertheless close in size (radius of gyration) to the native states of
the protein. The native contacts therefore remain relatively close to one another, which
may explain the apparent two-state cooperativity in the folding reactions in bulk
biochemistry experiments. Since both thermal and chemical perturbations drive the
proteins to very different initial unfolded states, it is dif
cult to examine the diversity of
folding reactions. Interestingly, even in the bulk some experiments have broadly
characterized the heterogeneity of the protein landscape, revealing that different
pathways become dominant depending on the folding conditions [9
18]. What is still
lacking is a consideration of the microscopic unfolding and refolding pathways under
the same external conditions to probe the dynamic diversity of the states exploredby the
same protein. Single-molecule AFM techniques are used to apply a denaturing force
along a well-de
ned reaction coordinate (end-to-end length) driving proteins to a fully
extended unfolded state. This level of control allows us to examine statistically the
folding pathways of the protein in question.
It should be noted that there is already a body of single-molecule literature showing
evidence of dynamic disorder onmultiple timescales that is explored by proteins and
enzymes in solution in their native states [19
-
23]. These results are obtained using
fluorescence and infrared spectroscopy to investigate the conformational changes of
the individual molecules over time, or the activity in enzymes. The observed complex
kinetics are signatures of multiple energy minima in the protein landscape, corre-
sponding to slightly different conformational sub-states of the molecule. The details
of the energy
fluctuations in the landscape can therefore be determined by following
each individual reaction by single-molecule experimentation and performing a
statistical analysis of the obtained distributions [24
-
29].
In this chapter we
first introduce the technique of force spectroscopy AFM and
the single-molecule
fingerprint achieved using polyprotein engineering. We then
present the recently developed constant force mode of the AFM (force-clamp
spectroscopy) as a tool to explore the force-dependent protein unfolding kinetics in
Section 13.3. A novel statistical analysis of such force-clamp data reveals deviations
from two-state kinetics and is discussed in terms of a disordered free energy
landscape in Section 13.4. Using this insightful technique in Section 13.5 we next
present protein refolding trajectories that reveal the complexity in the folding
pathways explored under a stretching force. We relate these results to simplistic
physical models of protein folding and uncover the need for the development of more
sophisticated theoretical frameworks for the understanding of this important process.
Finally, we expand the capabilities of the force-clamp technique to observe individual
chemical reactions in a single protein in Section 13.6, revealing both the effect of force
on chemical kinetics as well as the structure of the reaction transition state.
-
13.2
Single-protein AFM Techniques
The advent of single-protein Atomic Force Microscopy (AFM) techniques combined
with protein engineering techniques has made it possible, for the
first time, to
examine the mechanical properties of both native and engineered tandem modular
