| Kinesins are biological motors with the remarkable capability of coupling chemical energy from ATP hydrolysis into structural changes that generate force and directed motion along microtubules. Serving as a model protein, findings from single molecule kinesin biophysics provide general insights into mechanobiology. Kinesin comprises of 14 families that vary in structure, function and biophysical properties. The goal of this project is to elucidate details of the chemomechanical coordination between the two heads of the Kinesin-2 motor family, during their processive stepping and relate their in vitro biophysical properties to their in vivo behavior. Kinesin-2 motors are involved in intraflagellar transport as well as cytoplasmic transport of melanosomes.;Understanding protein behavior at the single molecule level requires developing techniques to image and manipulate them. In chapter 2, development of quantum dots as fluorescent tags to enhance spatial and temporal resolution of imaging is discussed. Kinesin-1 was tagged with biotinylated quantum dots and viewed using epifluorescence and total internal reflection fluorescence microscopy (TIRFM). Both techniques exhibited similar image resolution and the velocities and run lengths obtained compared favorably with values in literature.;Optical tweezers open avenues in mechanobiology by enabling the application of mechanical loads and manipulation of single molecules. An optical tweezer was constructed with back focal plane interferometry detection, and detailed calibration was performed. Preliminary data of motor stepping with 8 nm step sizes at low motor velocity was demonstrated. This optical trap was used to conduct bead assays at minimal loads to study the processivity and velocity of Kinesin-1 and Kinesin-2 motors. Mouse KIF3A/B was compared to homodimeric chimeras, KIF3A/A and KIF3B/B, and to conventional Kinesin-1 motors. At saturating ATP, KIF3A/B moved at 436 +/- 129 nm/s and the homodimers moved at similar speeds, while Kinesin-1 moved at 703 +/- 136 nm/s. The run lengths of all three KIF3 motors were approximately 600 nm, while the run length for Kinesin-1 was three-fold higher. When the ATP concentration was reduced from 1 mM down to 1 microM, Kinesin-1 run lengths were constant, consistent with previous reports. This implies that Kinesin-1 waits in the same chemomechanical state regardless of the time it takes for ATP to bind. In contrast, the run length of KIF3A/A increased nearly three-fold when the ATP was lowered from 1 mM to 1 microM. This implies that during the time the motor waits for ATP to bind at limiting ATP levels, the motor transitions to a different chemomechanical state, resulting in a lower probability of detachment following ATP binding. Stochastic simulations of motor stepping showed that at saturating concentrations, ATP binds to the motor while both heads are bound to the microtubule leading the motor to have a higher probability of detachment from the microtubule. Structural biology indicates that KIF3 has a 3-amino acid longer neck linker than Kinesin-1, which reduces the rearward strain on the leading head and causes absence of ATP gating. These results provide constraints on the Kinesin-2 mechanochemistry. |
