Bulletin of the American Physical Society
2006 APS March Meeting
Monday–Friday, March 13–17, 2006; Baltimore, MD
Session K26: Focus Session: Single Molecule Biophysics I |
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Sponsoring Units: DBP DCMP Chair: David Nelson, Harvard University Room: Baltimore Convention Center 323 |
Tuesday, March 14, 2006 2:30PM - 2:42PM |
K26.00001: Conformational Dynamics of Adenylate Kinase: The Effects of Temperature and Mutation on Friction, Memory, and Reactivity Lucas Watkins, Karl Duderstadt, Sucharita Bhattacharyya, Haw Yang Enzymes reside on a convoluted free energy surface. This free energy surface generates the conformational dynamics that control activity. We use single molecule F\"orster Resonance Energy Transfer measurements to study these conformational dynamics and the physics that underly them in a model enzyme, Adenylate Kinase (AK), which catalyzes the disproportionation of ADP into AMP and ATP. Our microscope records time-dependent single-molecule trajectories as a list of single photon arrival times. We treat the distance trajectory that generates this data as a manifestation of a many-dimensional Langevin equation, projected onto the coordinate defined by our two labeling sites. Using a likelihood-based approach, we can then directly extract the potential of mean force and the friction coefficient from the raw photon-by-photon trajectories. Temperature-dependent studies allow calculation of entropy and enthalpy profiles from the measured potentials of mean force, while mutants in functionally-important regions allow us to understand the role of individual residues in dynamics and catalysis. Ultimately, this newly-developed method allows us to begin to draw direct connections between structure, dynamics, and reactivity. [Preview Abstract] |
Tuesday, March 14, 2006 2:42PM - 2:54PM |
K26.00002: Single Human Plasma Fibrinogen Molecule Imaging by PEEM and AFM Xianhua Kong, Jacob Garguilo, Crissy Rhodes, Robert Nemanich Human Plasma Fibrinogen (HPF), which is a protein involved in thrombosis, was studied by photoelectron emission microscopy (PEEM) and Atomic Force Microscopy (AFM). PEEM, using the spontaneous emission output of the Duke OK-4 free electron laser (FEL), clearly reveals the trinodular structure of the single fibrinogen molecule absorbed on oxidized silicon substrates. Moreover, PEEM images excited using various wavelengths between 249 and 310 nm reveal ionization thresholds of 4.6 eV for HPF. In addition, hydrogen-terminated silicon (H-Si) substrates and oxidized silicon substrates serving as model hydrophobic and hydrophilic surfaces were used to investigate the absorption coverage difference of fibrinogen molecules using ambient AFM. The images show that the fibrinogn absorption on H-Si substrates is significantly greater than that on the oxidized substrates. [Preview Abstract] |
Tuesday, March 14, 2006 2:54PM - 3:06PM |
K26.00003: Probing the low-resolution dynamics of biopolymers under force Ruxandra Dima, Changbong Hyeon, D. Thirumalai Single molecule force experiments are a major source of information for probing the structure and the dynamics of biopolymers. While the response of a biopolymer to the application of force, which is readily obtained from the force versus the extension curve, provides a glimpse of the underlying energy landscape, the much more interesting molecular-level behavior is not as easy to extract. In this context, we introduce a minimal model suitable for simulating the dynamics under force of biomolecules for long time-scales and with modest computational expenses. We will show that forced unfolding pathways predicted using this minimal model on systems that are challenging to simulate with conventional methods correlate very well with the results of recent force-unfolding experiments. Our findings underline the importance of understanding the interplay between tension propagation and the molecular relaxation time in pulling experiments. [Preview Abstract] |
Tuesday, March 14, 2006 3:06PM - 3:18PM |
K26.00004: Motion of single MreB bacterial actin proteins in \textit{Caulobacter} show treadmilling in vivo W.E. Moerner, SoYeon Kim, Zemer Gitai, Anika Kinkhabwala, Harley McAdams, Lucy Shapiro Ensemble imaging of a bacterial actin homologue, the MreB protein, suggests that the MreB proteins form a dynamic filamentous spiral along the long axis of the cell in \textit{Caulobacter crescentus}. MreB contracts and expands along the cell axis and plays an important role in cell shape and polarity maintenance, as well as chromosome segregation and translocation of the origin of replication during cell division. In this study we investigated the real-time polymerization of MreB in \textit{Caulobacter crescentus} using single-molecule fluorescence imaging. With time-lapse imaging, polymerized MreB could be distinguished from cytoplasmic MreB monomers, because single monomeric MreB showed fast motion characteristic of Brownian diffusion, while single polymerized MreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer implies that treadmilling is the predominant mechanism in MreB filament formation. These single-molecule imaging experiments provide the first available information on the velocity of bacterial actin polymerization in a living cell. [Preview Abstract] |
Tuesday, March 14, 2006 3:18PM - 3:30PM |
K26.00005: Co-operative unfolding of protein domains Buddhapriya Chakrabarti, Tanniemola B. Liverpool, Alex J. Levine How well does the worm-like chain force extension curve fit single-molecule protein unfolding data? Careful analysis of dynamic force spectroscopy data for different proteins[1] suggests that the compliance of a protein is generically larger than that predicted by the worm-like chain model. We propose that the observed excess compliance is due to pre-transitional conformational rearrangements within the protein domain that occur before the more dramatic failure of the domain as a whole. Using a generalization of the formalism introduced by Evans and Ritchie[2], we study protein--unfolding kinetics in our model where these internal conformational rearrangements are represented by a number of interacting Ising-type variables, which cooperatively escape over a barrier to the unfolded state. From this model, we predict a relation between the statistics of the fluctuations of the peak domain--unfolding force and the deviations of the force extension curves from the worm-like chain prediction. We suggest that, by using this approach, one can extract further details on the domain--unfolding pathway from extant force spectroscopy data. \\ $[1]$ D. J. Brockwell (private communication). \\ $[2]$ E. Evans, and K. Ritchie, Biophys. J., {\bf 72} 1541 (1997). [Preview Abstract] |
Tuesday, March 14, 2006 3:30PM - 3:42PM |
K26.00006: Quantitative analysis of tethered particle motion Philip Nelson, Chiara Zurla, Darren Segall, Doriano Brogioli, Rob Phillips, David Dunlap, Laura Finzi Tethered particle motion (TPM) is a single-molecule technique that consists in tethering a bead to a slide through a DNA molecule. The Brownian amplitude of motion of the bead provides information about the conformational changes of the DNA molecule. We describe an improved experimental protocol, and a data analysis algorithm to extract quantitative conclusions from the data. We then apply a theoretical model for the statistics of the bead motion, which are quite different from those of a free polymer. Our experimental data for chain extension versus tether length are in good agreement with the model, showing that TPM is a useful tool for monitoring large conformational changes such as DNA looping. Moreover, we present the first experimental determination of the full probability distribution function of bead displacements, and find excellent agreement with theory over a range of tether lengths. Knowing this distribution a priori enhances our ability to extract events such as loop formation from observed time series. [Preview Abstract] |
Tuesday, March 14, 2006 3:42PM - 4:18PM |
K26.00007: The Role of Fluctuations in Enzymatic Activity Invited Speaker: In ``What is Life'', a set of lectures delivered in 1944, Erwin Schrodinger states ``{\ldots}from all we have learnt about the structure of living matter, we must be prepared to find it working in a manner that \textbf{cannot} be reduced to the ordinary laws of physics [not because] there is any `new force', {\ldots} but because the construction is different from anything we have yet tested in the physical laboratory.'' I will briefly discuss how this prediction fared 60 years after these lectures were given. With the advent of molecular, structural and single molecule biology, we are developing an increasingly mechanistic understanding of bio-molecular systems. The molecular machinery of life is imbedded in a viscous fluid where friction and thermal fluctuations are huge. In particular, studies of RNA enzymes such as the ribosome and other ribozymes show that their construction and operation \textit{is} different than human designed machines that work in an environment where fluctuations and dissipation or minimized. [Preview Abstract] |
Tuesday, March 14, 2006 4:18PM - 4:30PM |
K26.00008: Protein Unfolding Energy Determined by Jarzynski's Equality Ching-Hwa Kiang, Nolan Harris, Leiming Li, Yang Song, Wei Liao The dynamic response of single protein molecules to mechanical forces and the relation of dynamics to equilibrium properties of biomolecules has been a subject of intense recent study. Characterization of the fluctuations in these small systems plays an important role in successful application of Jarzynski's equality to determine equilibrium free energies from nonequilibrium measurements. Here we used the atomic force microscope to manipulate single titin I27 molecules to unfold the protein, and we have applied Jarzynski's equality to calculate the free energy landscape for stretching this heart muscle protein. [Preview Abstract] |
Tuesday, March 14, 2006 4:30PM - 4:42PM |
K26.00009: Protein folding in a force-clamp Marek Cieplak, Piotr Szymczak Kinetics of folding of a protein held in a force-clamp are compared to an unconstrained folding. The comparison is made within a simple topology-based dynamical model of ubiquitin. We demonstrate that the experimentally observed rapid changes in the end-to-end distance mirror microscopic events during folding. However, the folding scenarios in and out of the force-clamp are distinct. [Preview Abstract] |
Tuesday, March 14, 2006 4:42PM - 4:54PM |
K26.00010: Using a Microcantilever Array for Detecting DNA Melting Sibani Biswal, Digvijay Raorane, Alison Chaiken, Arun Majumdar Microcantilever based sensors translate changes in Gibbs free energy due to macromolecular interactions into mechanical responses. We utilize the microcantilevers to observe surface stress changes in response to thermal dehybridization of surface grafted double stranded DNA oligonicleotides. We begin by immobilizing and hybridizing 20, 25, and 30 base pair DNA strands. Once the cantilever is heated, the DNA undergoes a transition as the complementary strand melts which results a cantilever deflection change. This deflection is due to changes to the electrostatic, ionic, and hydration interaction forces between the remaining immobilized DNA strands. For example, using a 20mer DNA strand in a 50 mM PBS buffer, the cantilever deflection shows an abrupt discontinuity at T $\sim $ 39$^{o}$C. When the salt concentration is lowered to 25 mM, we see a shift in the discontinuity to a lower temperature, T $\sim $ 30$^{o}$C. We also observe that DNA strands grafted onto the cantilever melt at lower temperatures compared to bulk solution due to the interactions between neighboring strands and the surface. We are also probing how base mismatches affect the cantilever deflection. This new technique has allowed us to probe DNA melting dynamics and leads to a better understanding of the stability of DNA complexes on surfaces. [Preview Abstract] |
Tuesday, March 14, 2006 4:54PM - 5:06PM |
K26.00011: Exploring the Electrical Conductivity of Cytochrome P450 by Nano-Electrode and Conductive Atomic Force Microscopy Debin Li, Jianhua Gu, Yewhee Chye, David Lederman, Jarod Kabulski, Peter Gannett, Timothy Tracy There is a growing interest in measuring the conductivity of electron-transfer proteins. The cytochrome P450 (CP450) enzymes represent an important class of heme-containing enzymes. Immobilizing CP450 enzymes on a surface can be used for studying a single enzyme with respect to electron transfer. The spin state of the heme iron can change upon binding of a substrate. In our experiment, CP450 (diameter $\sim $ 5 nm) has been bonded to a metal surface. Nano-electrodes (gap $<$ 10 nm) were fabricated by defining a bridge via e-beam lithography and then breaking the junction by electromigration at low temperatures. We have examined the electronic properties of CP450 by itself and after binding CP450 with flurbiprofen. The room temperature I-V conductivity is reminiscent to cyclic voltammetry measurements, indicating the presence of strong ionic transfer. At lower temperatures (100 K) the I-V characteristics indicate electronic transport dominated by tunneling processes. The conductive AFM is an additional method used to examine the enzyme's electronic properties. The results from two methods will be discussed.. [Preview Abstract] |
Tuesday, March 14, 2006 5:06PM - 5:18PM |
K26.00012: Traveling wave tracking of individual molecular motors Irene Dujovne, M. van den Heuvel, C. Symonds, G. Cappello, Cees Dekker Insight into the mechanisms of motility can be obtained by the study of the movement of motor proteins along biological filaments. Optical tweezer techniques are now able to track the motion of motor proteins with nanometer spatial resolution and 1-10 Hz bandwidth. Recently, G. Cappello developed an optical technique to track the movement of beads with sub-nanometer and microsecond resolution $^1$. A unique aspect of this technique, based on total-internal-reflection traveling wave, is that no force is applied on the object under study. In this work we present modifications of this optical technique with the goal of larger scattering intensities that allow shorter acquisition times. Here we present this system to the study of microtubules traveling on kinesin-coated structures. The impact of fabricated nanostructures on the scattering intensities is discussed together with the feasibility of the application of this experimental scheme to the study of DNA-processing enzymes. \par \noindent 1 L. Busoni, A. Dornier, Jean-Louis Viovy, J.Prost, and G. Cappello, J. Appl. Phys. 98, 064302 (2005) [Preview Abstract] |
Tuesday, March 14, 2006 5:18PM - 5:30PM |
K26.00013: Steering and Trapping Multiple Particles by Feedback Flow Control: Theory and Experiments Mike Armani, Satej Chaudhary, Roland Probst, Benjamin Shapiro On the macro scale, feedback control is routinely applied to improve performance and enable new tasks in complex and uncertain systems operating in noisy environments. Our lab has focused on applying feedback control ideas to systems on the micro scale. We show how to combine micro-fluidics and feedback control to independently steer multiple particles with micrometer accuracy in two spatial dimensions. The particles are steered by creating a spatially and temporally varying fluid flow that carries all the particles from where they are to where they should be at each time step. Our control loop comprises sensing, computation, and actuation to steer particles along user-input trajectories, to hold particles in place, or both. Particle locations are identified in real-time by an optical system and sent to a control algorithm that then determines the electrode voltages necessary to create a flow field to carry all the particles to their next desired locations. The process repeats at the next time instant. We have demonstrated flow steering of multiple particles at once both in simulations and in experiments. The steering algorithm is robust to uncertainty and works even when conditions of the particles (size, surface charge), conditions of the buffer (pH, temperature, electro-chemistry, impurities), and attributes of the devices (errors in fabrication geometry, parasitic pressure flows driven by surface tension) vary and/or are unknown. [Preview Abstract] |
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