Measurement of Single Macromolecule Orientation by Total Internal Reflection Fluorescence Polarization Microscopy

Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania, Philadelphia, 19104-6083, USA.
Biophysical Journal (Impact Factor: 3.97). 09/2005; 89(2):1261-71. DOI: 10.1529/biophysj.104.053470
Source: PubMed


A new approach is presented for measuring the three-dimensional orientation of individual macromolecules using single molecule fluorescence polarization (SMFP) microscopy. The technique uses the unique polarizations of evanescent waves generated by total internal reflection to excite the dipole moment of individual fluorophores. To evaluate the new SMFP technique, single molecule orientation measurements from sparsely labeled F-actin are compared to ensemble-averaged orientation data from similarly prepared densely labeled F-actin. Standard deviations of the SMFP measurements taken at 40 ms time intervals indicate that the uncertainty for individual measurements of axial and azimuthal angles is approximately 10 degrees at 40 ms time resolution. Comparison with ensemble data shows there are no substantial systematic errors associated with the single molecule measurements. In addition to evaluating the technique, the data also provide a new measurement of the torsional rigidity of F-actin. These measurements support the smaller of two values of the torsional rigidity of F-actin previously reported.

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Available from: Margot E Quinlan, Apr 23, 2014
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    • "Figure 15.2 illustrates the benefit of our improved analysis in the context of polTIRF studies of myosin V; see Section 2.1. In the figure, the dots represent orientations determined by the traditional method by binning the data into 80 ms intervals (Forkey et al., 2005). Although they generally cluster around the results of our method (horizontal lines), the latter is cleaner and eliminates the spurious outlier points that the traditional analysis generates close to changepoints (Section 1.2). "
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    ABSTRACT: 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.
    Preview · Article · Jan 2011 · Methods in enzymology
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    • "With the current setup, the average orientation of the probe during each 80 ms detection cycle is resolved to within approximately 10°. Procedures for calibration and data collection can be found in Forkey et al., 2003; Quinlan et al., 2005; and Rosenberg et al., 2005. "
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    ABSTRACT: Myosin VI is an unconventional motor protein with unusual motility properties such as its direction of motion and path on actin and a large stride relative to its short lever arms. To understand these features, the rotational dynamics of the lever arm were studied by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy during processive motility of myosin VI along actin. The axial angle is distributed in two peaks, consistent with the hand-over-hand model. The changes in lever arm angles during discrete steps suggest that it exhibits large and variable tilting in the plane of actin and to the sides. These motions imply that, in addition to the previously suggested flexible tail domain, there is a compliant region between the motor domain and lever arm that allows myosin VI to accommodate the helical position of binding sites while taking variable step sizes along the actin filament.
    Full-text · Article · Jan 2008 · Molecular Cell
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