Single Molecule Force Spectroscopy using Optical Traps and AFMs

COFFEE_KLATCH · Invited

Abstract

Force spectroscopy is an important single-molecule technique to study the energetics and dynamics of biological systems. Both optical traps and atomic force microscopes (AFMs) can measure the dynamics of individual molecules. My talk will focus on two intellectually distinct ways to improve these experiments: passive force clamps and an optically stabilized AFM. To increase measurement precision, feedback is used to maintain a constant force on a molecule - often called a force clamp. Precise yet rapid active feedback is limited by Brownian motion. This limited bandwidth leads to significant fluctuations in force that are particularly pronounced for the rapid, large changes in extension seen in nucleic acid structures (e.g. DNA hairpins, ribozymes, riboswitches). Here, we show that the dynamics determined in active force clamps are five-to-seven fold different than in a passive force clamp, which has a ($\sim$30-fold faster control of force. Thus, the dynamics of biological molecules can be significantly altered by the mechanism of force feedback. In AFM-based force spectroscopy experiments, force versus extension curves are generated by retracting the tip using a PZT stage while measuring force via cantilever deflection. Extension is not stable over the long times due to drift in the AFM assembly ($\sim$10 nm/min). We developed an ultrastable AFM by measuring and thereby stabilizing the tip in 3D using a laser scattered off the apex of a commercial AFM tip, not its back side. A second laser detected and thereby stabilized the sample. We next demonstrated simultaneous and independent measurement of extension and force. Preliminary studies of bacteriorhodopsin, a model membrane protein, highlight this instruments unique force- and position-clamp modes.

–

Authors

  • John Poate

    Brigham Young University, Los Alamos National Laboratory, Department of Physics and Astronomy, University of Utah, USA, MV Systems, Inc., USA, Helmholtz-Zentrum Berlin fuer Materialien und Energie, Abteilung Silizium-Photovoltaik, Germany, Colorado School of Mines, Department of Physics, USA, Georgia Institute of Technology, Arizona State University, Physics Department of Babolsar University, Iran, Physics Department, New Mexico State University, Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, 85287-1604, USA, Department of Electrical Engineering, University of Arkansas, Fayetteville, AR 72701, USA, Department of Physics, Arizona State University, Tempe, AZ, 85287-1504, USA, Colorado State University, University of Wisconsin, NSF ERC for Extreme Ultraviolet Science and Technology, Colorado State University, BYU-Provo, Michigan Technical University and Pierre Auger Collaboration, University of Colorado, Colorado School of Mines, Department of Physics, Colorado State University, Department of Physics, Cornell University, NASA, University of Massachusetts at Amherst, University of Massachusetss at Amherst, APS President, Harvard University, Society of Physics Students, Duke University, Computer Science, Brigham Young University, Chemistry \& Biochemistry, Brigham Young University, University of Arizona, University of Utah, Kansas State Univ., Bethel University, University of New Mexico, Stanford University, JILA, University of Colorado at Boulder, NIST, JILA, University of Colorado at Boulder, National Renewable Energy Laboratory, University of Denver, University of Colorado, Boulder, NREL, Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, 85287- 1604, USA, DU, ERI, Eleanor Roosevelt Institute (ERI), Cerro Tololo Interamerican Observatory, Utah State University, Center for Atmospheric and Space Sciences, Sciprint.org, University of Colorado at Boulder, JILA and University of Colorado, Kirchhoff Institute for Physics, University of Heidelberg, Utah Valley University, University of New South Wales, San Francisco State University, Weber State University, Cambridge University, Department of Physics and Astronomy, University of Utah, Kansas State University, Columbia University, NY, University of Colorado/JILA, Vice-President for Research and Technology Transfer, Colorado School of Mines