Anthony Butterfield1, Russell Stewart2, Christoph Schmidt3, and Mikhail Skliar1. (1) Chemical Engineering, University of Utah, 50 S. Central Campus Dr., Salt Lake City, UT 84112, (2) Bioengineering, University of Utah, Salt Lake City, UT 84112, (3) Faculty of Physics, Georg-August-Universität of Göttingen, Göttingen, Germany
Details of force and movement production by molecular motors can be extracted from single-molecule recordings during optical trapping experiments. Previous analyses of single-molecule data for non-processive motors, such as myosin and ncd, used ensemble averaging to detect temporal structure within the power stroke these motors produce. Decrease in variance of measured displacement was used to mark the beginning and end of motor-filament attachment events. The mere decrease in variance, however, is insensitive to the type of event that causes it, and ensemble averaging obscures infrequent types of molecular events. We have developed a new analysis method that uses Kalman filtering of measurements, model-based estimation of the power strokes produced by the motor head, and automatic event classification to discriminate between different types of motor events. We applied the method to optical trap measurements of ncd power strokes in a three-bead geometry. In this configuration the power strokes produced by the motor head attached to a surface-immobilized bead causes the measured displacement of a single trapped bead suspending a microtubule above the motor. The examination of hundreds of identified events revealed that the majority was consistent with the previously discovered minus-end directed power stroke of ncd occurring at the end of the microtubule interaction. A minority of different types of events, however, were also present, namely apparent motor strokes in the plus-direction and binding events that did not terminate in a discernible power stroke. Automatic event classification was used to quantify the frequency of the different types of motor events. It was found that: (1) approximately 30% of all motor strokes were in the plus-direction; (2) approximately 6% of all microtubule-motor attachments ended without an identifiable motor stroke. Ensemble averaging was then used separately for each event category, which revealed that plus- and minus-directed strokes have different size and occur at different instants within the ncd-microtubule attachment sequence. Further details can be found at http://implicit.che.utah.edu/~misha/biophysj.pdf.