Abstract:
Single-molecule methods are promising to provide deep insight into all biological problems. Methods include among others single-molecule fluorescence and force measurements. Such measurements are limited by Brownian motion that puts bounds to the spatiotemporal resolution and by nonspecific interactions of the molecules of interest to probes and surfaces that are involved in the measurement. To reduce nonspecific interactions and improve the spatiotemporal resolution of single-molecule assays, I have developed several novel methods in this thesis. First, I have developed a highly reproducible supported solid lipid bilayer platform for such assays. This platform provides specific and load bearing attachments with less nonspecific interactions of biomolecules and probes to the surface. Thus, the lipid bilayer platform enabled fluorescence and force measurements to study the mechanics of single DNA molecules and molecular motors. One such molecular motor is kinesin that transports vesicles along microtubules. Kinesin motor proteins walk in a rotary hand-over-hand mechanism associated with a center-of-mass displacement of 8nm during the hydrolysis cycle. Early reports indicated that kinesin takes substeps. However, despite constant research over the last three decades, load-bearing substeps could not be confirmed and the stepping mechanism of kinesin remains unclear. An ideal tool, to study the mechanics of kinesin are optical tweezers. Optical tweezers use microspheres as handles for measuring piconewton forces generated by kinesin. However, micron-sized probes with a low refractive index limit the spatiotemporal resolution. To overcome this limit, I have synthesized high refractive index germanium nanospheres and antireflection coated, core-shell germanium microspheres. High-refractive index particles beyond a certain size limit cannot be trapped by a laser in optical tweezers unless they are photonically structured to reduce the scattering force. To reduce scattering and to increase the trap force, I added an antireflection coating to germanium microspheres. With these probes, we can, in the future, increase the optical force generated by the optical tweezers beyond a nanonewton or achieve a very fast response time below a microsecond. The germanium nanospheres already enabled me to perform high spatiotemporal optical tweezers measurements. I found that, instead of 8-nm steps, kinesin-1 motors take 4-nm center-of-mass steps with alternating step durations depending on force and ATP. At high loads, motors switched to a weakly bound state, but did not detach. Instead, motors slipped on microtubules in 8 nm steps on a microsecond time scale. Surprisingly, rescue of directed motion after such a slip event happened in about 80% of events. Such rescue events indicated that the run lengths of individual motors are concatenations and rescues need to be accounted for to understand long-range transport. By these measurements, I have resolved a longstanding controversy bringing together the kinesin stepping and detachment behavior. In sum, this thesis provides mechanistic insights into how kinesin-1 walks and detaches from microtubules, thus helps to understand the big picture of how kinesin motors work in a team to transport vesicles. In the long-term, employment of novel probes that enable an ultrahigh resolution opens up new avenues for detailed investigations and new discoveries of conformational changes that are key for the biological function of many other molecular machines.
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