SFB1032 Special Seminar

Venue: LMU Faculty of Physics, Altbau Physik, Kleiner Physikhörsaal (N020)

Host: Prof. Hermann Gaub (A01)

Probing protein folding using state-of-the-art atomic force microscopy 

Protein folding is understood as a set of transitions between states. Yet, an oversimplified
view of the folding process emerges if briefly populated states remain undetected due to
ensemble averaging and/or limited instrumental precision. Atomic force microscopy (AFM)-
based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure
the folding and unfolding of proteins. However, the temporal resolution of a standard
cantilever is 50–1,000 μs, masking rapid transitions and short-lived intermediates. Moreover,
equilibrium studies, where one measure a molecule as it repeatedly unfolds and refolds, has
not been achieved due to a lack of force stability. I will focus on a set of improvements to
AFM cantilevers that leads to enhanced force stability, force precision and temporal
resolution. Specifically, we demonstrated sub-pN force precision and stability over a broad
bandwidth (Δf = 0.01–20 Hz) by removing a cantilever’s gold coating. Next, we extended the
AFM’s sub-pN bandwidth by a factor of ~50 to span five decades of bandwidth (Δf = 0.01–
1,000 Hz). To do so, we used a focused ion-beam to micromachine a short (L = 40 μm)
commercial cantilever and then extended this concept further to modify an ultrashort
cantilever (L = 9 μm), achieving 1-μs temporal resolution on a commercial AFM. To
demonstrate the biophysical utility of these modified cantilevers, we unfolded a polyprotein,
a popular assay. In the second assay, we unfolded bacteriorhodopsin (bR), a model
membrane protein. The resulting force-extension curves for bR show unprecedented detail.
Numerous, newly detected intermediate states—many separated by as few as 2–3 amino
acids—exhibited complex dynamics, including frequent refolding transitions and state
occupancies of <10 μs. Equilibrium measurements between such states enabled the folding
energy landscape to be deduced. These results dramatically sharpen the picture of
membrane protein folding and, more broadly, enable experimental access to previously
obscured protein dynamics. I will conclude by by briefly discussing ongoing work in our lab that
focus on further improvements throughput and data quality.