Nelson, Philip C.

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Author: Beausang, John F ×

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Now showing 1 - 7 of 7
  • Publication
    Twirling of Actin by Myosins II and V Observed via Polarized TIRF in a Modified Gliding Assay
    (2008-12-01) Beausang, John F; Schroeder, Harry W; Nelson, Philip C; Goldman, Yale E
    The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay in order to measure the rotation of an actin filament about its axis (“twirling”) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeledactin monomers, using polarized total internal reflection (polTIRF) microscopy. In order to determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement four-fold. We found that whole myosin II and myosin V both twirl actin with a relatively long (~ µm), left-handed pitch that is insensitive to myosin concentration, filament length and filament velocity.
  • Publication
    Changepoint Analysis for Single-Molecule Polarized Total Internal Reflection Fluorescence Microscopy Experiments
    (2011-07-01) Goldman, Yale E; Beausang, John F; Nelson, Philip C
    The experimental study of individual macromolecules has opened a door to determining the details of their mechanochemical operation. Motor enzymes such as the myosin family have been particularly attractive targets for such study, in part because some of them are highly processive and their “product” is spatial motion. But single-molecule resolution comes with its own costs and limitations. Often, the observations rest on single fluorescent dye molecules, which emit a limited number of photons before photobleaching and are subject to complex internal dynamics. Thus, it is important to develop methods that extract the maximum useful information from a finite set of detected photons. We have extended an experimental technique, multiple polarization illumination in total internal reflection fluorescence microscopy (polTIRF), to record the arrival time and polarization state of each individual detected photon. We also extended an analysis technique, previously applied to FRET experiments, that optimally determines times of changes in photon emission rates. Combining these improvements allows us to identify the structural dynamics of a molecular motor (myosin V) with unprecedented detail and temporal resolution.
  • Publication
    DNA Looping Kinetics Analyzed Using Diffusive Hidden Markov Model
    (2007-01-31) Beausang, John F; Zurla, Chiara; Manzo, Carlo; Dunlap, David; Finzi, Laura; Nelson, Philip
    Tethered particle experiments use light microscopy to measure the position of a micrometer-sized bead tethered to a microscope slide via a micrometer length polymer, in order to infer the behavior of the invisible polymer. Currently, this method is used to measure rate constants of DNA loop formation and breakdown mediated by repressor protein that binds to the DNA. We report a new technique for measuring these rates using a modified hidden Markov analysis that directly incorporates the diffusive motion of the bead, which is an inherent complication of tethered particle motion because it occurs on a time scale between the sampling frequency and the looping time. We compare looping lifetimes found with our method, which are consistent over a range of sampling frequencies, to those obtained via the traditional threshold-crossing analysis, which vary depending on how the raw data are filtered in the time domain. Our method does not involve such filtering, and so can detect short-lived looping events and sudden changes in looping behavior.
  • Publication
    Tethered Particle Motion as a Diagnostic of DNA Tether Length
    (2006-08-01) Nelson, Philip C; Zurla, Chiara; Brogioli, Doriano; Beausang, John F; Finzi, Laura; Dunlap, David
    The tethered particle motion (TPM) technique involves an analysis of the Brownian motion of a bead tethered to a slide by a single DNA molecule. We describe an improved experimental protocol with which to form the tethers, an algorithm for analyzing bead motion visualized using differential interference contrast microscopy, and a physical model with which we have successfully simulated such DNA tethers. Both experiment and theory show that the statistics of the bead motion are quite different from those of a free semiflexible polymer. Our experimental data for chain extension versus tether length fit our model over a range of tether lengths from 109 to 3477 base pairs, using a value for the DNA persistence length that is consistent with those obtained under similar solution conditions by other methods. Moreover, we present the first experimental determination of the full probability distribution function of bead displacements and find excellent agreement with our theoretical prediction. Our results show that TPM is a useful tool for monitoring large conformational changes such as DNA looping.
  • Publication
    Elementary Simulation of Tethered Brownian Motion
    (2007-01-26) Beausang, John F; Zurla, Chiara; Finzi, Laura; Sullivan, Luke; Nelson, Philip C.
    We describe a simple simulation, suitable for an undergraduate project or graduate problem set, of the Brownian motion of a particle in a Hooke’s law potential well. Understanding this physical situation is necessary in many experimental contexts, for instance in single molecule biophysics, and its simulation helps students appreciate the dynamical character of thermal equilibrium. The simulation captures behavior seen in experimental data on tethered particle motion.
  • Publication
    Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence Microscopy
    (2013-03-01) Beausang, John F; Shroder, Deborah Y; Nelson, Philip C; Goldman, Yale E
    Myosin V is biomolecular motor with two actin-binding domains (heads) that take multiple steps along actin by a hand-over-hand mechanism. We used high-speed polarized total internal reflection fluorescence (polTIRF) microscopy to study the structural dynamics of single myosin V molecules that had been labeled with bifunctional rhodamine linked to one of the calmodulins along the lever arm. With the use of time-correlated single-photon counting technology, the temporal resolution of the polTIRF microscope was improved ~50-fold relative to earlier studies, and a maximum-likelihood, multitrace change-point algorithm was used to objectively determine the times when structural changes occurred. Short-lived substeps that displayed an abrupt increase in rotational mobility were detected during stepping, likely corresponding to random thermal fluctuations of the stepping head while it searched for its next actin-binding site. Thus, myosin V harnesses its fluctuating environment to extend its reach. Additional, less frequent angle changes, probably not directly associated with steps, were detected in both leading and trailing heads. The high-speed polTIRF method and change-point analysis may be applicable to single-molecule studies of other biological systems.
  • Publication
    Calibration of Tethered Particle Motion Experiments
    (2009-07-01) Han, Lin; Lui, Bertrand H; Blumberg, Seth; Beausang, John F; Nelson, Philip C; Phillips, Rob
    The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA loping and transcription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead’s random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better characterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200 bp to 2.6 kbp and consider particle diameters of 200, 490 and 970 nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation.