Goldman, Yale E.

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Now showing 1 - 5 of 5
  • 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
    Self-Assembled Charged Hydrogels Control the Alignment of Filamentous Actin
    (2009-01-01) Park, Jung Hyun Hyun; Goldman, Yale E.; Composto, Russell J; Sun, Yujie
    We demonstrate a novel route to control attachment of filamentous actin (F-actin) on hydrogel films. By incorporating an amine-terminated silane, the hydrogel surface charge and surface topography are varied. With increasing silane content, F-actin reorients from perpendicular to parallel to the hydrogel surface, ceases to wobble, and forms mainly elongated or cyclic structures. F-Actin coverage reaches a maximum at 2.5 vol% silane and declines at higher silane content. This biphasic behavior is explained by the simultaneous increase in surface charge and the self-assembly of a micron scale pattern of positively charged islands. Our approach provides guidelines for constructing nanoscale tracks to guide motor proteins underlying nano-engineered devices such as molecular shuttles.
  • Publication
    Using electrical and optical tweezers to facilitate studies of molecular motors
    (2009-03-09) Sun, Yujie; Bau, Haim H.; Arsenault, Mark E.; Goldman, Yale E.
    Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells.
  • Publication
    Confinement and Manipulation of Actin Filaments by Electric Fields
    (2007-08-01) Purohit, Prashant K; Arsenault, Mark E; Goodman, Yale E; Zhao, Hui; Bau, Haim H
    When an AC electric field was applied across a small gap between two metal electrodes elevated above a surface, rhodamine-phalloidin-labeled actin filaments were attracted to the gap and became suspended between the two electrodes. The variance 〈s2(x)〉 of each filament's horizontal, lateral displacement was measured as a function of electric field intensity and position along the filament. 〈s2(x)〉 markedly decreased as the electric field intensity increased. Hypothesizing that the electric field induces tension in the filament, we estimated the tension using a linear, Brownian dynamic model. Our experimental method provides a novel means for trapping and manipulating biological filaments and for probing the surface conductance and mechanical properties of single polymers.
  • Publication
    The mechanics of short rod-like molecules in tension
    (2008-05-01) Purohit, Prashant K; Goldman, Yale; Bau, Haim H; Arsenault, Mark E
    The rapid development of single molecule experimental techniques in the last two decades has made it possible to accurately measure the force-extension response as well as the transverse fluctuations of individual rod-like macromolecules. This information is used in conjunction with a statistical mechanical model based on the treatment of the molecule as a fluctuating elastic rod to extract its bending and extension moduli. The models most commonly used to interpret the experimental data assume that the magnitude of the Brownian fluctuations are independent of the length of the macromolecule, an assumption that holds only in the asymptotic limit of infinitely long rods, and is violated in most experiments. As an alternative, we present a theoretical treatment of a finite length, fluctuating rod and determine its mechanical behavior by measuring the transverse Brownian fluctuations under the action of large stretching forces. to validate of our theory, we have applied our methods to an experiment on short actin filaments whose force-extension relation is difficult to measure, but whose transverse deflections can be captured by current microscopy techniques. An important consequence of the short contour lengths is that the boundary conditions applied in the experiment affect the fluctuations and can no longer be neglected as is commonly done when interpreting data from force-extension measurements. Our theoretical methods account for boundary conditions and can therefore be deployed in conjunction with force extension measurements to obtain detailed information about the mechanical response of rod-like macromolecules.