Nanoscale single-molecule tracking with thin films and reflected light

University Of California Santa Barbara, Santa Barbara CA

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Abstract

DESCRIPTION (provided by applicant): Many critical cellular processes, such as DNA replication and chromosome segregation, are carried out by the activity of DNA based motor proteins. These enzymes transduce chemical energy into physical motion, such as the unzipping of the double helix, or the transfer of chromosomes within the cell. Recent technical developments have allowed quantitative real time tracking of the motion of single motor proteins, and consequent deduction of the structural and biochemical mechanisms of their activity. However, this work has been hampered by a lack of experimental precision: DNA based motor proteins are thought to move forward by one to a few basepairs per enzymatic cycle (: per step :), which lies below the spatial resolution of most single molecule tracking techniques. Thus, current techniques cannot directly measure the size of individual steps, which are of paramount importance when attempting to determine a protein9s mechanism. Determination of the microscopic mechanisms of motor protein activity is key to understanding how they carry out their tasks, including those whose malfunction lead to disease. We propose to rectify this situation by developing a single molecule measurement apparatus with high spatial resolution. The apparatus will be based on the magnetic tweezers;an experimental setup in which the motion of a DNA tethered magnetic bead reflects the motion of a motor protein. In order to increase the resolution of the magnetic tweezers, we will apply a highly sensitive reflective interference technique. Further, the quality of the interference pattern will be enhanced by fabricating thin films of gold or high dielectric material on the relevant interfaces;the design of those thin films will be based on ray tracing simulations. With these changes, we estimate that the instrument will have a total resolution of 0.2 nm at the data acquisition rate of 60 Hz. Small amounts of low pass filtering will improve this resolution: e.g. using a 30 Hz bandwidth, the resolution will be 0.1 nm. This improves magnetic tweezers resolution 5 10 fold over traditional methods;more importantly, it brings the resolution into the sub basepair regime needed to track the stepping of DNA based motor proteins. Finally, we expect that, once developed, this combination of a high resolution measurement in a relatively simple instrument will find applications to other biomolecular systems. Narrative Proteins are the machines of the cell: they use energy in order to carry out tasks such as copying, repairing, and moving DNA. The precise manner in which they perform their tasks is a medically important subject, since misdirected or missing protein activity can lead to cell malfunction, cell death, and disease. In order to learn more about the precise nature of protein mechanisms, we propose to develop techniques that allow protein activity to be tracked with unprecedented spatial resolution.