ABSTRACT

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 ;10! 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.

INTRODUCTION

High-resolution structures of proteins trapped in distinct static configurations have shown that rotational motions of compact domains are a common feature of their enzymatic and energy transducing mechanisms (1–11). In all of these systems, plausible relationships have been proposed between the observed rotational motions and the functional output, but testing the structurally derived hypotheses requires detection of the timing and extent of the rotations during activity.

Fluorescence polarization on bulk biological samples, such as suspensions of macromolecules or lipid vesicles, is a commonly used method to detect rotational motions (12,13). The signals are typically sensitive to the time course and extent of motions of the probe molecules. The method is well suited to detect time-resolved structural changes in organized systems, such as proteins embedded in lipid membranes (14) or muscle fibers (15). In these samples having a symmetry axis, the absolute distribution of probe orientations relative to that axis becomes available (16). With the recent availability of probes having known local orientation within the domain of interest (17,18), the information can be directly converted into distributions and motions of the local molecular domain. Among techniques available for detecting protein rotational motions, fluorescence polarization has the advantages of good sensitivity and time resolution and being relatively straightforward to set up and interpret.

In bulk experiments, however, relating the information about protein distributions to the orientation and dynamics of individual protein molecules is difficult because of averaging over an unsynchronized population. Abrupt perturbations of a molecular ensemble, such as temperature or pressure jumps, or rapid addition of substrate, can partially synchronize the population, but some important characteristics, such as fast or backward reaction steps and rare states, are usually not accessible.

Single molecule fluorescence measurements have been used to avoid the complications caused by the ensemble averaging in measurements on molecular distributions.Whereas most single molecule experiments have been focused on detecting the temporally resolved location of individual molecules, some have used fluorescence polarization to also determine structural information (e.g., 19–22). Single molecule fluorescence polarization has the potential to bridge the gap between techniques with atomic spatial resolution but poor time resolution (e.g., x-ray crystallography) and those capable of resolving kinetics but giving somewhat ambiguous structural information (e.g., fluorescence energy transfer).

Here, we present single molecule fluorescence polarization instrumentation that utilizes total internal reflection (TIR) microscopy. The TIR excitation offers an advantage over epifluorescence and confocal microscopy of a strong polarization component along the z axis (optical axis of the microscope) as well as along the x and y axes, making it possible to determine the three-dimensional (3D) orientation (22,23). Analytical techniques are described to determine, with a temporal resolution of ;1–40 ms, the full 3D orientation of individual fluorescent probes and the extent of their wobbling motions on the microsecond and subnanosecond timescales. Comparison of single molecule and ensemble measurements of rhodamine bound to actin filaments are

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