Changepoint Analysis for Single-Molecule Polarized Total Internal Reflection Fluorescence Microscopy Experiments

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.

Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence Microscopy

“…In a previous study (31) we developed a multiple-channel change-point (MCCP) algorithm tailored to single-photon polTIRF experiments. In this algorithm, a likelihood function is used to compare two hypotheses: 1), the hypothesis that there is a single, abrupt intensity change at the i th photon in an interval containing N photons in time T; and 2), the null hypothesis that there is no intensity change at the i th photon.…”

Maximum-likelihood approach to topological charge fluctuations in lattice gauge theory

“…36.1.2 of [8]) can produce reliable estimates of model parameters when there are relatively few independent samples of a distribution, provided the functional form of the underlying distribution is known analytically. Inspired by the work of Phil Nelson and collaborators [9], we present such a maximum likelihood approach (following an example by Franco [10] in the context of financial time-series) to analyze the complete time-series information {Q n }. The analysis is done without blocking, since the effect of autocorrelations is built into the model.…”

Elongation factor G initiates translocation through a power stroke

“…1 C-E). A maximumlikelihood, multitrace change-point algorithm identifies statistically valid change points caused by sudden rotations and localizes their timing within several milliseconds (26). For each instance of EF-G BR binding to the ribosome, the orientation and extent of wobble of EF-G BR before and after any structural changes were determined from the PFIs (details in Supporting Information).…”