Real-Time Single Protein Tracking with Polarization Readout using a Confocal Microscope

Abstract

DNA binding proteins are very efficient in recognizing/searching target sites over long stretches of non-specific DNA sequences. Current theories suggests that the search efficiency comes partly from non-specific interactions with DNA. During these DNA interactions the search dimensionality is lowered from a three-dimensional diffusion process to a one-dimensional. When this occurs the protein is for a short time sliding along the DNA contour. Indirect evidence suggest that the protein is rotating along the DNA spiral but the underlying dynamics of these sliding events are still not well understood. To experimentally investigate sliding dynamics, over time scales comparable to expected protein rotation times, we have developed a single molecule tracking polarization microscope. The platform is based on the scanning confocal principle and has capability of real time positioning feedback, polarization measurement and single photon time tagging. Photon acquisition and positioning controls are all implemented on an FPGA allowing for fast feedback without losing single photon timing information. With a reaction time close to a millisecond, for the positioning feedback, and a 200 Mhz clock for time tagging we study sliding of the lac repressor. Our lac repressors are each labeled with a fluorophore that is predicted to be in a rigid conformation. This allows us to use polarization as an indicator of the protein orientation. Using flow stretched DNA we are able to capture DNA-protein sliding events. Preliminary results reveals a polarization shift that is consistent with the expected dipole orientation and flow direction and thus support the rotational hypothesis.