Bulletin of the American Physical Society
APS March Meeting 2010
Volume 55, Number 2
Monday–Friday, March 15–19, 2010; Portland, Oregon
Session A10: Focus Session: Single Molecule Biophysics and Chemical Physics I |
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Sponsoring Units: DCP DBP DPOLY Chair: David Rueda, Wayne State University Room: A106 |
Monday, March 15, 2010 8:00AM - 8:36AM |
A10.00001: Single-molecule studies of multi-protein machines Invited Speaker: Advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. In a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. Our group is developing the single-molecule tools that will make it possible to study biochemical pathways of arbitrary complexity at the single-molecule level. I will discuss results of single-molecule experiments on the replisome, the molecular machinery that is responsible for replication of DNA. We stretch individual DNA molecules and use their elastic properties to obtain dynamic information on the proteins that unwind the double helix and copy its genetic information. Furthermore, we visualize fluorescently labeled components of the replisome and thus obtain information on stochiometry and exchange kinetics. This simultaneous observation of catalytic activity and composition allows us to gain deeper insight into the structure-function relationship of the replisome. [Preview Abstract] |
Monday, March 15, 2010 8:36AM - 8:48AM |
A10.00002: Single-Molecule Measurements of Synthesis by DNA Polymerase with Base-Pair Resolution Thomas Christian, Louis Romano, David Rueda The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3'-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of \textit{E. coli} DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule F\"{o}rster resonance energy transfer intensity drops in discrete steps to values consistent with single nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer-template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change following the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the pre-insertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process. [Preview Abstract] |
Monday, March 15, 2010 8:48AM - 9:00AM |
A10.00003: Dissecting the dissociation process of RecA monomer from a nucleoprotein filament Doseok Kim, Sung Hyun Kim, Chirlmin Joo, Jeehae Park, Kaushik Ragunathan, Taekjip Ha RecA protein is a DNA-dependent ATPase and plays a key role in DNA repair mechanisms. RecA proteins form a helical filament on a single-strand DNA mediating homologous recombination. Understanding the molecular mechanisms of RecA-DNA interaction is crucial for further investigation on its biochemical properties. Using a single-molecule fluorescence technique, we dissected the dissociation process with single-monomer resolution. We could resolve the existence of an intermediate state after ATP hydrolysis in the dissociation process. In the nucleotide cofactor free environment, RecA did not dissociate indicating that the bound ADP is required for the monomer dissociation. Based on our observation, we suggest a model for the RecA dissociation process coupled with ATP hydrolysis cycle. [Preview Abstract] |
Monday, March 15, 2010 9:00AM - 9:12AM |
A10.00004: Dynamics of DNA strand exchange by Bxb1 integrase, a model serine site-specific recombinase Hua Bai, Pallavi Ghosh, Mingxuan Sun, Nigel Grindley, Graham Hatfull, John F. Marko Site-specific recombination breaks and rejoins DNA at specific sequences within synaptic complexes assembled by specialized recombinase enzymes. Structural data suggest that serine recombinases exchange duplex DNAs via a ``clutch plate'' mechanism allowing the fully cleaved duplex DNA ends to be exchanged by a rigid body rotation. We have directly observed this rotational motion for a simple serine recombinase, the Bxb1 phage integrase, using a single-DNA supercoiling-release assay which allows us to follow cleavage, rotation, religation and product release in real time. The molecular friction associated with the bearing is much larger than that found for type I topoisomerases in a similar assay. Experiments with recombination-incompetent and recombination-competent substrates lead to expected outcomes. We confirm our results in two-DNA braiding-relaxation experiments where synapse rotation can be directly observed in reactions on two long molecules. [Preview Abstract] |
Monday, March 15, 2010 9:12AM - 9:24AM |
A10.00005: Noise Induced Regulation of DNA Loops Joshua Milstein, Yih-Fan Chen, Jens-Christian Meiners Protein-mediated DNA loop formation arises from thermal fluctuations within a crowded and active intracellular environment. Constant forces, on the order of Femtonewtons, have been shown to modulate the rate of loop formation, which suggests that tension may act to regulate genetic transcription. To understand how such a mechanism might operate within the cellular environment, we have been exploring the effects of a fluctuating force on the formation and dissociation rates of LacI-mediated DNA loops. Employing axial constant-force optical tweezers, we apply white noise Gaussian fluctuations, of varying widths and at different mean applied forces, to a length of dsDNA containing two \textit{lac} binding sites while observing the looping dynamics. Our empirical observations, in conjunction with a stochastic model of the loop formation process, suggest that applying a varying level of tension to the DNA may, in fact, be a robust method for regulating transcription. [Preview Abstract] |
Monday, March 15, 2010 9:24AM - 9:36AM |
A10.00006: DNA Micromanipulation Using Novel High-Force, In-Plane Magnetic Tweezer Christopher McAndrew, Patrick Mehl, Abhijit Sarkar We report the development of a magnetic force transducer that can apply piconewton forces on single DNA molecules in the focus plane allowing continuous high precision tethered-bead tracking. The DNA constructs, proteins, and buffer are introduced into a 200$\mu $L closed cell created using two glass slides separated by rigid spacers interspersed within a thin viscoelastic perimeter wall. This closed cell configuration isolates our sample and produces low-noise force-extension measurements. Specially-drawn micropipettes are used for capturing the polystyrene bead, pulling on the magnetic sphere, introducing proteins of interest, and maintaining flow. Various high-precision micromanipulators allow us to move pipettes and stage as required. The polystyrene bead is first grabbed, and held using suction; then the magnetic particle at the other end of the DNA is pulled by a force created by either two small (1mm x 2mm x 4mm) bar magnets or a micro magnet-tipped pipette. Changes in the end-to-end length of the DNA are observable in real time. We will present force extension data obtained using the magnetic tweezer. [Preview Abstract] |
Monday, March 15, 2010 9:36AM - 9:48AM |
A10.00007: Complex kinetics of the $\lambda $ Bacteriophage genetic switch Laura Finzi, Carlo Manzo, Chiara Zurla, David Dunlap The kinetics of the $\lambda $ bacteriophage repressor-mediated DNA loop formation and breakdown were characterized by Tethered Particle Microscopy (TPM). A generalized likelihood ratio test was first applied to determine the location of change points (cp) in the TPM trace. Expectation-maximization (EM) clustering and the Bayesian information criterion were then used for accurate determination of the number of states accessible to the system. This procedure (cp-EM) allows objective and quantitative determination of TPM change points without the artificial time resolution limitations that arise from filtering and thresholding. Only two states were identified, which corresponded to the looped to the unlooped DNA configurations. The probability distribution function of the looped and unlooped DNA state dwell times revealed a complex kinetics. In particular, it was found that a stretched exponential provided a satisfactory fitting for the probability distribution of the unlooped state dwell times, while the dwell time distribution for the looped DNA state could not be fitted with a standard pdf; we observed, however, that a power law decay fits well the long dwell times. A mechanism is proposed to explain this kinetic behavior, where $\lambda $ repressor non-specific binding to DNA may play an important physiological role. [Preview Abstract] |
Monday, March 15, 2010 9:48AM - 10:00AM |
A10.00008: ABSTRACT WITHDRAWN |
Monday, March 15, 2010 10:00AM - 10:12AM |
A10.00009: Detailed Computational Study of Translocation Dynamics: Revealing the Physical Mechanisms of Viscosity Dependent Scaling Laws Hendrick W. de Haan, Gary W. Slater Noting the limitations of current methods of characterizing the unbiased translocation of a polymer through a nanopore, we demonstrate a measurement which more completely maps out the process in time. Applying this approach to Langevin Dynamics simulations of translocation at different viscosities for a relatively tight nanopore yields interesting results for the scaling of the translocation time with polymer length: $\tau \sim N^\alpha$. At low viscosities, super-diffusive results are obtained. At high viscosities, while translocation is found to obey simple scaling with $\alpha=2.2$ near the beginning of the process, length dependent deviations from simple scaling near the end result in an overall $\alpha$ exponent which is viscosity dependent. The memory and entropic effects giving rise to this behaviour will be discussed. [Preview Abstract] |
Monday, March 15, 2010 10:12AM - 10:24AM |
A10.00010: Shape transition in channel- and cavity-confined semiflexible bio-macromolecules Peter Cifra, Tomas Bleha Stiff macromolecules entrapped in channels or spherical cavities undergo a shape transition on increasing confinement as shown by our investigation using molecular simulations. In channels this weak-to-strong confinement transition leads to extended conformations without the hairpin-like back-folding. In cavities, on decrease of cavity radius, the semiflexible chain in a disordered state starts to organize into the torus. This happens when the extent of confinement reaches the lower bound of macromolecular flexibility given by the minimal radius of chain curvature or the persistence length. As a common rule for both types of confinement the transition to the ordered structures is observed when the radius of cavity or cylindrical channel comes down to the persistence length of macromolecular chain. This simple geometric rule finds its application in various confinement situations of stiff bio-macromolecules either in micro channel experiments or real biophysical situation such as DNA in viral capsids. [Preview Abstract] |
Monday, March 15, 2010 10:24AM - 10:36AM |
A10.00011: Out of Equilibrium Characteristics of a Forced Translocating Chain through a Nanopore Kurt Binder, Aniket Bhattacharya Polymer translocation through a nano-pore in a thin membrane is studied using Langevin dynamics simulation with a particular emphasis to explore out of equilibrium characteristics of the translocating chain. We analyze the chain conformations both at the $cis$ and the $trans$ side separately. A detail picture of translocation emerges by monitoring the center of mass of the translocating chain, longitudinal and transverse components of the gyration radii and the end-to-end vector. We observe that polymer configurations at the $cis$ side are distinctly different from those at the $trans$ side. During the translocation, and immediately afterwards, different parts of the chain are characterized by a series of effective Flory exponents. We further notice that immediately after the translocation the last set of beads that have just translocated take a relatively compact structure compared to the first set of beads that translocated earlier and the translocation dynamics can be described as a propagating defect. We discuss implications of these results to the theoretical estimates and numerical simulation studies of the translocation exponent reported by various groups. [Preview Abstract] |
Monday, March 15, 2010 10:36AM - 10:48AM |
A10.00012: ABSTRACT WITHDRAWN |
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