Bulletin of the American Physical Society
Fall 2009 Meeting of the Four Corners Section of the APS
Volume 54, Number 14
Friday–Saturday, October 23–24, 2009; Golden, Colorado
Session B2: Symposium on Soft Matter: Biophysics I |
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Chair: Sean Shaheen, University of Denver Room: Green Center 210S |
Friday, October 23, 2009 2:10PM - 2:34PM |
B2.00001: Single Molecule Force Spectroscopy using Optical Traps and AFMs Invited Speaker: 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. [Preview Abstract] |
Friday, October 23, 2009 2:34PM - 2:46PM |
B2.00002: Induction of Electrode-Cellular Interfaces with $\sim $ 0.05 $\mu $m$^{2}$ Contact Areas Bret Flanders, Prem Thapa Individual cells of the slime mold \textit{Dictyostelium discoideum} attach themselves to negatively biased nanoelectrodes that are separated by 30 $\mu $m from grounded electrodes. There is a -43 mV voltage-threshold for cell-to-electrode attachment, with negligible probability across the 0 to -38 mV range but probability that approaches 0.7 across the -46 to -100 mV range. A cell initiates contact by extending a pseudopod to the electrode and maintains contact until the voltage is turned off. Scanning electron micrographs of these interfaces show the contact areas to be of the order of 0.05 $\mu $m$^{2}$. Insight into this straight-forward, reproducible process may lead to new electrode-cellular attachment strategies that complement established approaches, such as blind sampling and patch clamp. [Preview Abstract] |
Friday, October 23, 2009 2:46PM - 2:58PM |
B2.00003: Probing Kv2.1 Channel Dynamics Using Single Molecule Tracking Aubrey Weigel, Michael Tamkun, Diego Krapf Kv2.1 potassium channels localize into micron-sized clusters in live neurons. This exceptional characteristic is essential for cellular function. Nevertheless, the physical mechanism behind Kv2.1 cluster formation and maintenance is largely unknown. We are investigating the dynamics of clustered Kv2.1 channels using total internal reflection fluorescence microscopy to track single molecules with nanometer accuracy in real time. Human embryonic kidney (HEK) cells are employed as a model system. HEK cells are induced to express biotinylated Kv2.1 channels fused to green fluorescent protein (GFP). Single channels are detected with streptavidin-conjugated red quantum dots (QD). GFP fluorescence provides characteristics of clusters as an ensemble while the red QDs enable tracking of individual channels. We study the dynamics of single channels inside the clusters and at the cluster interface in terms of their mean square displacement (MSD) and cumulative distribution function. Our results show a bimodal distribution of channels (clustered and non-clustered) and indicate that both Kv2.1 populations experience anomalous subdiffusion independent of cluster perimeter. [Preview Abstract] |
Friday, October 23, 2009 2:58PM - 3:10PM |
B2.00004: Force-activated reactivity switch in a bimolecular chemical reaction at the single molecule level Robert Szoszkiewicz, Sergi Garcia-Manyes, Jian Liang, Tzu-Ling Kuo, Julio M. Fernandez Mechanical force is a distinct and usually less explored way to activate chemical reaction because it can deform the reacting molecules along a well-defined direction of the reaction coordinate. However, the effect of mechanical force on the free- energy surface that governs a chemical reaction is still largely unknown. The combination of protein engineering with single-molecule force-clamp spectroscopy allows us to study the influence of mechanical force on the rate at which a protein disulfide bond is reduced by some reducing agents in a bimolecular substitution reaction (so-called SN2). We found that cleavage of a protein disulfide bond by hydroxide anions exhibits an abrupt reactivity ``switch'' at 500 pN, after which the accelerating effect of force on the rate of an SN2 chemical reaction greatly diminishes. We propose that an abrupt force- induced conformational change of the protein disulfide bond shifts its ground state, drastically changing its reactivity in SN2 chemical reactions. Our experiments directly demonstrate the action of a force-activated switch in the chemical reactivity of a single molecule. References: S. Garcia-Manyes, J. Liang, R. Szoszkiewicz, T-L. Kuo and J. M. Fernandez, Nature Chemistry, 1, 236-242, 2009. [Preview Abstract] |
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