MEMS Seminar: Improving the Time Resolution and Force Precision of bioAFM Reveals a Multitude of Hidden Dynamics in the Unfolding of Membrane Protein

Sep 13

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Wednesday, September 13, 2017 - 12:00pm to 1:00pm


Professor Thomas Perkins

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) enables a wide array of studies, from measuring the strength of a ligand-receptor bond to elucidating the complex folding pathway of individual membrane proteins. Such SMFS studies and, more generally, the diverse applications of AFM across biophysics and nanotechnology are improved by enhancing the force stability, force precision and time resolution of bioAFM. For an advanced, small-format commercial AFM, we uncovered how these three metrics were limited by the cantilever itself rather than the larger microscope structure, and then describe three increasingly sophisticated cantilever modifications that yield enhanced data quality. First, sub-pN force precision and stability over a broad bandwidth (Δf = 0.01–20 Hz) is routinely achieved by removing a long (L = 100 μm) cantilever’s gold coating. Next, this sub-pN bandwidth is extended by a factor of 50 to span five decades of bandwidth (Δf = 0.01–1000 Hz) by using a focused ion

beam (FIB) to modify a shorter (L = 40 μm) cantilever. Finally, FIB-modifying an ultrashort (L = 9 μm) cantilever improves its force stability and precision while maintaining 1-μs temporal resolution. We then applied these technological improvements to reexamine the unfolding of a model membrane protein, bacteriorhodopsin, with a 100-fold improvement in time resolution and a 10-fold improvement in force precision. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates—many separated by as few as 2–3 amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 µs. Equilibrium measurements between such states enabled the underlying folding free-energy landscape to be deduced. Finally, recent efforts to improve data quantity and data accuracy of AFM-based SMFS studies will be discussed.

Thomas Perkins is a Fellow and Associate Chair of JILA, a staff member of NIST’s Quantum Physics Division, and a Professor (adjoint) of the Molecular, Cellular and Developmental Biology Department at the University of Colorado. He graduated from Harvard University, magna cum laude in physics. Tom’s graduate work under the guidance of Steve Chu at Stanford focused on developing single-molecule techniques to study issues in polymer physics He did his postdoctoral training with Steve Block at both Princeton and Stanford in high-resolution studies of DNA-based molecular motors. Tom’s group specializes in developing and applying high-precision single-molecule techniques, such as atomic force microscopy and optical traps to answer biological questions. Tom has received the Padden Award for outstanding polymer physics thesis, a Burroughs Wellcome Fund Career Award in the Biomedical Sciences, the Marinus Smith Award for significant impact on lives of CU undergraduate students, and the Arthur S. Flemming Award for outstanding achievement in government service.

Lunch will be served at 11:30 am.