47 research outputs found
Myosin VI Lever Arm Rotation: Fixed or Variable?
Get PDFTwo recent articles addressed the power-stroke of myosin VI molecules during stepping. Although both groups measured the angles of fluorescent probes attached on the myosin VI molecule lever arm using polarized fluorescence techniques, they differ about whether the myosin VI lever arm rotation is fixed1 or variable2. Here we discuss the causes of the discrepancy between the two studies and the implications for myosin VI processive motility
Single molecule investigations of DNA looping using the tethered particle method and translocation by acto-myosin using polarized total internal reflection fluorescence microscopy
Get PDFSingle molecule biophysics aims to understand biological processes by studying them at the single molecule level in real time. The proteins and nucleic acids under investigation typically exist in an aqueous environment within ∼ ten degrees of room temperature. These seemingly benign conditions are actually quite chaotic at the nanoscale, where single bio-molecules perform their function. As a result, sensitive experiments and statistical analyses are required to separate the weak single molecule signal from its background. Protein-DNA interactions were investigated by monitoring DNA looping events in tethered particle experiments. A new analysis technique, called the Diffusive hidden Markov method, was developed to extract kinetic rate constants from experimental data without any filtering of the raw data; a common step that improves the signal to noise ratio, but at the expense of lower time resolution. In the second system, translocation of the molecular motor myosin along its actin filament track was studied using polarized total internal reflection (polTIRF) microscopy, a technique that determines the orientation and wobble of a single fluorophore attached to the bio-molecule of interest. The range of resolvable angles was increased 4-fold to include a hemisphere of possible orientations. As a result, the handedness of actin filament twirling as it translocated along a myosin-coated surface was determined to be left-handed. The maximum time resolution of a polTIRF setup was increased 50-fold, in part by recording the arrival times and polarization state of single photons using a modified time-correlated single photon counting device. A new analysis, the Multiple Intensity Change Point algorithm, was developed to detect changes in molecular orientation and wobble using the raw time-stamped data with no user-defined bins or thresholds. The analysis objectively identified changes in the orientation of a bifunctional-rhodamine labeled calmodulin that was attached to a myosin V molecule translocating along an actin filament. Long intervals corresponding to stable positions between tilting motions of the lever arm during each step were routinely observed. Substeps in the cycle that preceded these long dwells were measured, but only occasionally most likely because of the low number of photons detected during these rapid events
Calibration of Tethered Particle Motion Experiments
Get PDFThe 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
The Symphonies of John Kinsella
Get PDFSéamas de Barra
The Symphonies of John Kinsella
ABSTRACT
This thesis offers the first comprehensive analytical and critical study of the symphonies of John Kinsella (b. 1932), one of the leading figures in contemporary Irish music. This cycle of ten works represents the most substantial contribution to the genre by an Irish composer, and Kinsella’s varied handling to the form is examined and discussed in relation both to historical and contemporary developments. While his understanding of musical structure and the manner in which he shapes musical time are deeply indebted to the work of Jean Sibelius, Kinsella’s compositional idiom is derived from a personal adaptation of serialism in which the technique of the note-row is manipulated to readmit the forces of tonal attraction. The result of these twin influences is an arrestingly individual approach to composition, the development of which is traced across the cycle as each of the symphonies in turn is subjected to extensive analysis. Because he chose to pursue an independent path in the 1980s, Kinsella seemed a somewhat isolated figure to his contemporaries. Retrospectively, his work can be seen as instinctively in tune with broader developments, however, as both serialism (understood as a way of thinking rather than as a style) and the music of Sibelius have emerged as two of the dominant influences on current musical thinking
DNA Looping Kinetics Analyzed Using Diffusive Hidden Markov Model
Get PDFTethered 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
Changepoint Analysis for Single-Molecule Polarized Total Internal Reflection Fluorescence Microscopy Experiments
Get PDFThe 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
Frequency Selective Surfaces as Near Infrared Electro-Magnetic Filters for Thermophotovoltaic Spectral Control
Get PDFFrequency selective surfaces (FSS) effectively filter electromagnetic radiation in the microwave band (1 mm to 100 mm). Interest exists in extending this technology to the near infrared (1 {micro}m to 10 {micro}m) for use as a filter of thermal radiation in thermophotovoltaic (TPV) direct energy conversion. This paper assesses the ability of FSS to meet the strict spectral performance requirements of a TPV system. Inherent parasitic absorption, which is the result of the induced currents in the FSS metallization, is identified as a significant obstacle to achieving high spectral performance
Twirling of Actin by Myosins II and V Observed via Polarized TIRF in a Modified Gliding Assay
Get PDFThe 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
Elementary Simulation of Tethered Brownian Motion
Get PDFWe 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
