Our protocol is designed to probe the biophysical mechanisms governing the transport of cargo by teams of identical or oppositely directed cytoskeletal motor proteins. By using DNA origami for molecular construction, this method can choose for the type, number, and location of motors on precisely defined cargo shapes. While this protocol focuses on the microtubule-based motors dynein and kinesin, it is adaptable to the actin-based motor myosin, as well as to other protein systems that function cooperatively.
One challenging aspect of this protocol is to maintain the integrity and activity of the motor proteins throughout the purification process, as they're sensitive to heat and mechanical force. To induce motor protein growth and expression via galactose promoter, use a sterile inoculation wand to streak the frozen yeast strain of interest on a yeast-peptone-dextrose culture plate for a three-to four-day incubation at 30 degrees Celsius. On day four of culture, when the optical density at 600 nanometers is between 1.5 and two, collect the yeast cells by centrifugation, and resuspend the pellet in water to wash them and consolidate the cells in a single bottle.
Spin the cells again for collection, and resuspend the pellet in about two milliliters of double-distilled water. Then use a 10-milliliter pipette to slowly dispense the cell slurry into liquid nitrogen one drop at a time to produce frozen yeast cell pellets. For motor protein purification, use a blade-type coffee grinder pre-chilled with liquid nitrogen to grind the frozen yeast pellets into a fine powder, and transfer the yeast powder into a pre-chilled, 100-milliliter, glass beaker on ice.
Add a small volume of freshly prepared 4X lysis buffer with supplements to the powder so that the final concentration of the buffer does not exceed 1X, and place the beaker in a 37-degree Celsius water bath with continuous stirring with a spatula to quickly thaw the powder. Add additional 4X buffer with supplements to bring the final buffer concentration to 1X in the lysate. The final volume is often between 25 and 30 milliliters.
Then collect the yeast cells by centrifugation, and transfer the soluble protein containing supernatant into a new 50-milliliter conical tube on ice. For IgG affinity purification, add 200 microliter of washed affinity bead suspension to the protein extract, and incubate the mixture at four degrees Celsius for one hour with gentle rotation. At the end of the incubation, filter the mixture through the same chromatography column used to prepare the beads at four degrees Celsius, followed by two washes with five milliliters of wash buffer per wash on ice.
After the second wash, rinse the beads on ice with five milliliters of TEV buffer, allowing the buffer to fully drain from the column. For DNA oligonucleotide labeling, cap the chromatography column, and incubate the motor beads with 100 microliters of TEV buffer supplemented with 10 to 20 micromolar of the purified benzylguanine oligo at room temperature for 10 to 15 minutes, gently resuspending the beads once a minute. At the end of the incubation, wash the beads four times with fresh TEV buffer per wash as just demonstrated, before recapping the bottom of the tube to allow resuspension of the motor beads in no more than 200 microliters of fresh TEV buffer.
For TEV cleavage, transfer the motor bead suspension to a two-milliliter, round-bottom, microcentrifuge tube, and incubate the motor beads with approximately 0.3 units of TEV protease per microliter of motor bead mixture at 16 degrees Celsius for one hour with gentle rotation. At the end of the incubation, centrifuge the motor beads, and collect the mixture at the bottom of the tube. Next, use a cut P1000 pipette tip to transfer the mixture to a spin column, and centrifuge the mixture as just demonstrated.
Collect the filtrate containing the TEV-cleaved, purified motors, and aliquot the filtrate in 50-microliter volumes for microtubule affinity purification. Then flash freeze the aliquots in liquid nitrogen for minus 80-degree Celsius storage. For chassis purification, in the late afternoon of the day before the purification, gently layer 80 microliters each of concentration of glycerol in origami folding buffer in a centrifuge tube.
The boundaries between the layers should be slightly visible. After an overnight incubation at four degrees Celsius, layer 10%glycerol in origami folding buffer in folded chassis solution over the top layer of the gradient, and centrifuge the gradient with the chassis. At the end of the centrifugation, collect 50-microliter fractions of the gradient in a top-to-bottom direction, and load five microliters of each fraction into individual wells of a 2%agarose gel.
After 90 to 120 minutes at 70 volts, image the gel using conditions suitable for the DNA gel stain used. Quantified concentrations of the selected fractions of interest can be determined using the appropriate standard spectroscopic methods. SDS-PAGE analysis can be used to confirm the successful extraction of dynein from yeast, as the final filtrate shows a clear, sharp band at approximately 350 kilodaltons.
TEV protease is also present in the final filtrate and forms a clear band at about 50 kilodaltons. After microtubule affinity purification, dynein appears as a clear, single band at 350 kilodaltons, while kinesin can be observed as slightly smearing, multiple bands at about 120 kilodaltons. In addition to the TEV protease, a noticeable amount of tubulin can also be observed in the final supernatant.
The folding of DNA origami structures can be assayed by agarose gel electrophoresis, with a shift in mobility between the pure unfolded scaffold strand and the folding reaction indicating origami folding. Additionally, the folding reaction indicates the presence of some multimerization of chassis structures. The motility of motor chassis ensembles is easily detectable and measurable on kymographs generated from TIRF movies, as the kymographs of flexible chassis conjugated to seven dynein proteins demonstrate highly processive runs at relatively consistent velocities.
It is vital to carefully control the temperature of the motor proteins at each step of the procedure. Following the purification of the motor proteins and DNA origami chassis, the two components can be conjugated for motility assays of the ensembles under a TIRF microscope. This method enables the biophysical and biochemical mechanisms that govern the emergent motility of the motor ensembles to be deciphered.
