I am currently using atomic force microscopy (AFM) to measure the energetics stabilizing membrane proteins, especially the model membrane protein bacteriorhodopsin. In my first published contribution to this area, I developed a new technique (dubbed "zigzag force spectroscopy") to extract more usable information from each individual molecule probed compared to traditional techniques.
"Membrane-protein unfolding intermediates detected with enhanced precision using a zigzag force ramp"
(Featured as a "New and Notable" article in Biophysical Journal)
With Hao Yu, I then used AFM-based unfolding to measure the unfolding free energy of an eight-amino-acid region of bacteriorhodopsin, overcoming the limitations present when such a measurement is made using the traditional approach of chemical denaturation in detergent micelles.
"Quantifying the native energetics stabilizing bacteriorhodopsin by single-molecule force spectroscopy"
In the course of this work, I developed an analytical theory for correcting unfolding/refolding kinetic rates in cases where the AFM time resolution is limiting.
"Correcting molecular transition rates measured by single-molecule force spectroscopy for limited temporal resolution"
I then extended this approach to measure the change in unfolding free energy when a single-amino-acid point mutation is introduced. This reports on the contribution of the mutated amino acid's side chain to the overall stability of the protein. Using single-molecule techniques opens an avenue for making such measurements even when traditional biochemical assays cannot be applied.
"Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy"
Left: Illustration of an experiment in which mechanical force is used to unfold bacteriorhodopsin. The force needed to disrupt the native structure reports on the underlying stability of that structure.