The Perkins Laboratory: Single Molecule Biophysics

Tom Perkins and Allison Churnside in the laboratory.

Single molecule studies are revolutionizing biophysics. One outstanding question is how motor proteins transduce chemical energy into physical motion? Another is how does the structure and dynamics of membrane proteins affect their functions? The Perkins group focuses on developing precision single-molecule techniques and applying them to answer these and other interesting biological questions.

One single-molecule technique that we are exploiting is the optical trap (or optical tweezers). Borrowing concepts from AMO physics, we developed an optical-trapping assay with Ångström-scale stability and resolution in all three dimensions (3D). Briefly, we minimize various types of laser noise (pointing, mode, polarization, and intensity) to achieve Ångström-scale sensitivity. For surface-coupled single-molecule assays, we stabilize the sample in 3D to mitigate environmental-induced mechanical perturbations. We are currently applying these techniques to study of the mechanical properties of DNA, Dye-DNA interactions, DNA-based molecular motors, biologically derived force standards, and the folding/unfolding kinetics of RNA structures. We also continue to pursue advances in optical-trapping technology.

In a parallel effort, we developed an ultrastable atomic force microscope (AFM). The instrument is based on optical detection technology using back-scattered light, rather than the forward-scattered light commonly used in optical traps. We used this technique to measure and thereby control a microscope cover slip in 3D to less than1 Å. Next, we scatter a second laser off the apex-not the backside-of a commercial AFM tip. We can control an AFM tip to <0.4 Å in 3D. We integrated these two advances to achieve an ultrastable AFM. Specifically, we demonstrated an AFM that is a 100-fold more stable than the prior state of the art at biologically useful conditions. We are pursuing applications to imaging and mechanically unfolding important biological molecules, such as membrane proteins, as well as enhancements to the underlying technology. Applications of the ultrastable AFM to nanotechnology are also being pursued.