Bulletin of the American Physical Society
APS March Meeting 2011
Volume 56, Number 1
Monday–Friday, March 21–25, 2011; Dallas, Texas
Session L39: Focus Session: Single Molecule Biophysics III: Novel Single Molecule Approaches to Biology |
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Sponsoring Units: DBP DPOLY DCP Chair: Ching Hwa Kiang, Rice University Room: A124/127 |
Tuesday, March 22, 2011 2:30PM - 3:06PM |
L39.00001: High-resolution laser-based detection for magnetic tweezers Invited Speaker: Magnetic tweezers are a versatile and powerful single-molecule manipulation technique capable of applying force and torque on single bio-molecules. They afford several unique advantages over other single-molecule manipulation techniques such as optical tweezers or atomic force microscopy. The hallmark of magnetic tweezers is the ability to twist bio-molecules without the need for complex optical instrumentation. Perhaps less known but of equal significance, magnetic tweezers rely on a slowly decaying magnetic field gradient (1 mm) to impose force so they are intrinsically configured in a passive force clamp mode. These features make magnetic tweezers particularly well suited for the study of nucleic acid structure, DNA topology, and protein-nucleic acid interactions. The one downside to most magnetic tweezers to date is that they rely on video tracking methods to determine the position of the particle. Despite recent progress, the spatial and temporal resolution and accuracy are fundamentally limited by image tracking techniques. I will describe recent improvements utilizing laser-based detection to overcome these limitations. We implemented back-scattered laser-based detection combined with video image tracking to achieve high-resolution, high-bandwidth, three-dimensional position tracking. [Preview Abstract] |
Tuesday, March 22, 2011 3:06PM - 3:18PM |
L39.00002: Massively Parallel Single-Molecule Manipulation Using Centrifugal Force Wesley Wong, Ken Halvorsen Precise manipulation of single molecules has led to remarkable insights in physics, chemistry, biology, and medicine. However, two issues that have impeded the widespread adoption of these techniques are equipment cost and the laborious nature of making measurements one molecule at a time. To meet these challenges, we have developed an approach that enables massively parallel single- molecule force measurements using centrifugal force [1]. This approach is realized in the centrifuge force microscope, an instrument in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force- field while their micro-to-nanoscopic motions are observed. We demonstrate high- throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Currently, we are taking steps to integrate high-resolution detection, fluorescence, temperature control and a greater dynamic range in force. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.\\[4pt] [1] K. Halvorsen, W.P. Wong, Biophysical Journal - Letters 98 (11), (2010). [Preview Abstract] |
Tuesday, March 22, 2011 3:18PM - 3:30PM |
L39.00003: Horizontal Magnetic Tweezers for Micromanipulation of Single DNA-Protein Complexes C. McAndrew, A. Sarkar, P. Mehl We report on the development of a new magnetic force transducer or ``tweezer'' that can apply pico-Newton forces on single DNA molecules in the focus plane. Since the changes in DNA's end-to-end extension are coplanar with the pulling force, there is no need to continually refocus. The DNA constructs ($\lambda $-DNA end labeled with a 3$\mu $m polystyrene bead and a 2.8$\mu $m paramagnetic sphere) and appropriate buffer are introduced to a custom built 400$\mu $L to 650$\mu $L closed cell. This closed cell isolates our sample and produces low-noise force and extension measurements. This chamber rests on a stage fixed to a three axis micromanipulator. Entering the flat chamber are two micropipettes, a 2.5$\mu $m id pipette for aspirating the polystyrene bead and a 20$\mu $m id pipette for injecting proteins of interest. The suction and the injection pipettes are rigidly mounted to a hydraulic, three-axis micromanipulator. DNA-bead constructs, once introduced to the chamber, can be located by moving the stage over the objective. We have shown that we can easily and reputably find, capture, and manipulate single molecules of DNA within a force range of 0.1pN to 100pN. [Preview Abstract] |
Tuesday, March 22, 2011 3:30PM - 3:42PM |
L39.00004: Modeling the effects of internal and external fluctuations on lifetimes of proteins measured by an AFM Eric Corwin, Maxime Clusel Measurements of the distribution of the time to unfold a single- molecule of a given protein under an externally applied force have emerged as an important tool with which to study the mechanical stability and energy landscape of a protein. In such an experiment the protein is potentially subject both to internal fluctuations in structure as well as external fluctuations in temperature and applied force. We report on a theoretical exploration of the effects that each kind of fluctuation may have on the measured lifetime distribution. We show that it is extremely difficult to distinguish internal fluctuations from external fluctuations in the lifetime distribution. We find that the rate distribution has higher sensitivity to the origins of fluctuations. Therefore, we propose an experimental protocol to estimate the approximate magnitude of internal fluctuations by intentionally adding increasing amounts of external fluctuations and measuring the skewness of the resulting rate distribution. [Preview Abstract] |
Tuesday, March 22, 2011 3:42PM - 3:54PM |
L39.00005: Onset of excluded volume in poly(ethylene oxide) elasticity measurements Andrew Dittmore, Dustin B. McIntosh, Omar A. Saleh We use magnetic tweezers to control tension in an 80 kDa poly(ethylene oxide) (PEG) chain. In good solvent, force effectively transforms the swollen coil into a series of smaller polymers ("tension blobs") and progressively diminishes self-avoidance interactions between distant parts of the chain. Excluded volume effects dominate the low-strain elasticity, where the extension follows a 2/3 power law in force in accordance with scaling predictions. These effects disappear as the polymer first enters a linear power-law regime, and then a high-force asymptotic regime well described by the Marko-Siggia wormlike chain model. All told, we observe two transitions between three elastic regimes. We show that the transition forces can be used to determine the polymer's Kuhn length, excluded volume, and thermal blob size, and find that PEG requires roughly 30 Kuhn lengths before self-avoidance becomes significant. Thus, we show that single-molecule elasticity can quantify the onset of a polymer's excluded volume, a problem that has eluded bulk measurement techniques. [Preview Abstract] |
Tuesday, March 22, 2011 3:54PM - 4:06PM |
L39.00006: Peptide Nucleic Acids as Tools for Single-Molecule Sequence Detection and Manipulation Hagar Zohar, Craig Hetherington, Carlos Bustamante, Susan Muller The ability to strongly and sequence-specifically attach modifications such as fluorophores and haptens to individual double-stranded (ds) DNA molecules is critical to a variety of single-molecule experiments. We propose using modified peptide nucleic acids (PNAs) for this purpose and implement them in two model single-molecule experiments where individual DNA molecules are manipulated via microfluidic flow and optical tweezers, respectively. We demonstrate that PNAs are versatile and robust sequence-specific tethers. [Preview Abstract] |
Tuesday, March 22, 2011 4:06PM - 4:18PM |
L39.00007: Resolving Single Molecule Lysozyme Dynamics with a Carbon Nanotube Electronic Circuit Yongki Choi, Issa S. Moody, Israel Perez, Tatyana Sheps, Gregory A. Weiss, Philip G. Collins High resolution, real-time monitoring of a single lysozyme molecule is demonstrated by fabricating nanoscale electronic devices based on single-walled carbon nanotubes (SWCNT). In this sensor platform, a biomolecule of interest is attached to a single SWCNT device. The electrical conductance transduces chemical events with single molecule sensitivity and 10 microsecond resolution. In this work, enzymatic turnover by lysozyme is investigated, because the mechanistic details for its processivity and dynamics remain incompletely understood. Stochastically distributed binding events between a lysozyme and its binding substrate, peptidoglycan, are monitored via the sensor conductance. Furthermore, the magnitude and repetition rate of these events varies with pH and the presence of inhibitors or denaturation agents. Changes in the conductance signal are analyzed in terms of lysozyme's internal hinge motion, binding events, and enzymatic processing. [Preview Abstract] |
Tuesday, March 22, 2011 4:18PM - 4:30PM |
L39.00008: Mechanical properties of NRR domain from human Notch 1 studied by single molecule AFM force spectroscopy Robert Szoszkiewicz, Ashim Dey For proteins in living cells, forces are present from macroscopic to single molecule levels. Single molecule atomic force microscopy in force extension (FX-AFM) mode measures forces at which proteins undergo major conformational transitions with $\sim$10 pN force sensitivity (FX-AFM). Here, we present the results of the FX-AFM experiments on a construct comprising the NRR domain from human Notch 1. It is believed that understanding the mechanical properties of Notch at the single molecule level can help to understand its role in triggering some breast cancers. The experimental results on the Notch construct and further analysis revealed several conformational transitions of this molecule under force. These results opened a path for further investigations of Notch constructs at various physiologically relevant conditions. [Preview Abstract] |
Tuesday, March 22, 2011 4:30PM - 4:42PM |
L39.00009: Non-Perturbative Tracking of Processive DNA Synthesis with Single-Molecule Fluorescence Everett Lipman, Charles Wickersham We have demonstrated recently that double-stranded DNA labeled with a periodic series of fluorescent dyes can be used to track a single helicase. Here we describe how this technique can be modified to follow DNA synthesis. By means of a stepwise loss of fluorescence during strand displacement, we monitor processive motion of a single $\phi 29$ DNA polymerase without labeling or altering the enzyme or the template strand, and without applying any force. We observe a wide range of speeds, with the highest exceeding by several times that observed in other single-molecule experiments. Because this method enables repeated observations of the same polymerase traversing identical segments of DNA, it should prove useful for studying sequence-specific effects in DNA replication and transcription. [Preview Abstract] |
Tuesday, March 22, 2011 4:42PM - 4:54PM |
L39.00010: Single molecule studies reveal new mechanisms for microtubule severing Jennifer Ross, Juan Daniel Diaz-Valencia, Margaret Morelli, Dong Zhang, David Sharp Microtubule-severing enzymes are hexameric complexes made from monomeric enzyme subunits that remove tubulin dimers from the microtubule lattice. Severing proteins are known to remodel the cytoskeleton during interphase and mitosis, and are required in proper axon morphology and mammalian bone and cartilage development. We have performed the first single molecule imaging to determine where and how severing enzymes act to cut microtubules. We have focused on the original member of the group, katanin, and the newest member, fidgetin to compare their biophysical activities in vitro. We find that, as expected, severing proteins localize to areas of activity. Interestingly, the association is very brief: they do not stay bound nor do they bind cooperatively at active sites. The association duration changes with the nucleotide content, implying that the state in the catalytic cycle dictates binding affinity with the microtubule. We also discovered that, at lower concentrations, both katanin and fidgetin can depolymerize taxol-stabilized microtubules by removing terminal dimers. These studies reveal the physical regulation schemes to control severing activity in cells, and ultimately regulate cytoskeletal architecture. [Preview Abstract] |
Tuesday, March 22, 2011 4:54PM - 5:06PM |
L39.00011: Polymer Nanocomposites as a Facile Method for Engineering Acto-Myosin Networks at the Interface Matthew Caporizzo, Yujie Sun, Yale Goldman, Russell Composto Filamentous actin acts as the rails for the molecular motor myosin in muscle contraction and intercellular mass transport. Consequently, understanding the process by which actin organizes, polymerizes, and binds is fundamental for the design of myosin based actuators capable of responding to external stimuli. Starting with atomically smooth, freshly cleaved mica optically coupled to glass slides, a random copolymer nanoparticle composite is engineered for \textit{in situ} single molecule TIRF/AFM studies with controlled roughness, electrostatic binding strength, and binding site density. Four distinct regimes of actin binding are observed; no attachment, end-on attachment, weak side-on attachment, and side-on immobilization. Transitions between regimes are likely to mark competition between the affinity to charged nanoparticles and the inherent resistance of the semi-rigid filaments to bending. Surface conditions optimal for actin immobilization are identified, and Myosin V stepping kinetics are studied on the artificially immobilized filaments, confirming filament support of motility. Supported by NSF grant DMR-0425780. [Preview Abstract] |
Tuesday, March 22, 2011 5:06PM - 5:18PM |
L39.00012: Electrostatic Effects on the Elasticity of Single ssDNA Molecules Dustin B. McIntosh, Omar A. Saleh Nucleic acids are highly-charged polyelectrolytes whose structure and function strongly depend on the concentration and type of salt ions in solution. We have created a simple experimental system for studying nucleic acid/ ion interactions, based on magnetic-tweezer measurements of the elasticity of single denatured ssDNA molecules in solutions with a known salt concentration. Using this system, we were able to reconcile single-molecule force-extension data with scaling theories of self-avoiding polymers, and we found that the Kuhn length of ssDNA scales with the Debye length in NaCl solutions. (Saleh et al., PRL 102, 068301 (2009)). Here, we use the system to investigate interactions of ssDNA with multivalent salts. We find that, in divalent salt, ssDNA elasticity is qualitatively similar to that in monovalent salt, but with significant quantitative differences. Notably, at low ionic strength, ssDNA in divalent salt maintains the same low-force scaling behavior (``Pincus blob'' regime) as seen in monovalent salts. However, there are differences in the elastic behavior at high forces ($>$ a few pN). In addition, analysis of the low-force scaling behavior indicates it requires $\sim$100 fold smaller concentrations of divalent salt to condense ssDNA. We discuss the data in the context of electrostatic theories, including Debye-Huckel, as well as bulk experiments on similar systems. [Preview Abstract] |
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