Department of Physics Papers

Document Type

Journal Article

Date of this Version

7-2011

Publication Source

Methods Enzymol

Volume

487

Start Page

431

Last Page

463

DOI

10.1016/B978-0-12-381270-4.00015-9

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.

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Date Posted: 07 April 2017

This document has been peer reviewed.