Final Report Summary - NANOMECH (Protein Friction of Molecular Machines: Nanomechanics with Optical Tweezers)
The central aim of this project is to understand the mechanical working principles of molecular machines using optical tweezers, a ultra-sensitive force and position transducer. While protein components of many biological machines have been identified, and in many cases their structures have been solved, the mechanical principles that govern the operation of biological machines are poorly understood. For example, how much force can they generate; and what limits their speed and efficiency? We have developed optimized optical tweezers and novel high-refractive index, anti-reflection coated microspheres used as probes for the optical tweezers. With these probes, we have pushed the upper force limit of optical tweezers beyond a nanonewton. An increased force range enables novel experiments in physics and biology. For example, we could verify that the noise that drives Brownian motion is not white but colored, i.e. it has a frequency-dependent power spectral density. Or, we could show that a single-strand annealing protein strongly clamps DNA to detect and secure homology. We have applied the optical tweezers technology, to address how molecular machines interact with microtubule cytoskeletal filaments. To this end, we investigated kinesin-8 and other motor proteins that regulate microtubule length. We could show that kinesin-8 is the weakest kinesin motor characterized so far. The motor is not optimized for cargo transport, but to regulate the dynamics of microtubules at their ends. To reach these ends, the motor has a weakly-bound slip state acting as a molecular safety leash. In addition, the motor is able to switch microtubule protofilaments in a diffusive manner to efficiently bypass obstacles. Apart from active motor proteins, microtubule length is also regulated by catalytic enzymes. For the polymerase XMAP215 that enhances microtubule growth, we could show that a tensile force applied to XMAP215-coated microspheres increases microtubule growth speed. Thus, tensile force, possibly acting on XMAP215 during cell division, may regulate microtubule dynamics and thereby mitotic processes. Both the weakly-bound slip state of kinesin-8 and the state, in which XMAP215 targets microtubule ends, are diffusive states. If such states are biased, friction arises between the proteins. Thus, a mechanical characterization of these molecules gives insight on the molecular origin of protein friction and how it limits the efficiency of the molecular machines.