Session B14: Molecular Machines

Experiments on single-molecular energetics of biological molecular motor

A biological molecular motor is a nano-sized chemical engine that converts chemical free energy to mechanical motions. Hence, the elucidation of the energetics is of critical importance to understand its operation principle. We experimentally evaluated the thermodynamic properties of a rotational F₁-ATPase motor (F₁-motor) at a single-molecule level. For this purpose, we used nonequilibrium relations. We show that the F₁-motor achieves 100% thermodynamic efficiency at the stalled state. As well, the motor reduces the irreversible internal heat to almost zero during rotations far from a quasistatic process. We discuss F₁-motor's free-energy transduction mechanism, which highlights the remarkable property of the nano-sized engine F₁-motor. Furthermore, we found that the F₁-motor implements a rectification mechanism, which reduces futile energy consumption. The rectification mechanism is simple but circumvents the trade-off between the forward speed and forward/backward contrast suffered by a simple ratcheting mechanism. If time allows, I will also introduce our challenge to realize a synthetic molecular motor based on what we learned from the biological molecular motors. We are developing a DNA-origami-based motor with conformational change.   

The lawnmower: an artificial protein-based burnt-bridge molecular motor

Molecular motors are protein-based machines essential for directional transport of cellular components. Inspired by biology, we have synthesized and characterized a protein-based microscale motor we dub the lawnmower. It is comprised of a spherical hub decorated with trypsin enzymes, and its motion is directed by cleavage of a peptide lawn, which promotes motion towards fresh substrate. Thus, it is designed to act as a burnt-bridge ratchet. We find the lawnmower is capable of superdiffusive motion, and can attain average speeds of up to 80 nm/s. By contrast, lawnmowers exhibit exhibit purely diffusive motion on lawns lacking the peptide substrate. We believe the lawnmower is the first example of an autonomous protein-based synthetic motor purpose-built using nonmotor protein components.   

Single-molecule studies of KIF1A motion and force generation

The kinesin-3 motor KIF1A functions in neurons, where its fast and superprocessive motility facilitates long-distance transport, but little is known about its force-generating properties. Using optical tweezers, we demonstrate that KIF1A stalls at an opposing load of ~3 pN but more frequently detaches at lower forces. KIF1A rapidly reattaches to the microtubule to resume motion due to its class-specific K-loop, resulting in a unique clustering of force generation events. To test the importance of neck linker docking in KIF1A force generation, we introduced the V8M and Y89D disease mutations. Both mutations dramatically reduce the force generation of KIF1A but not the motor’s ability to rapidly reattach to the microtubule. Although both mutations relieve autoinhibition of the full-length motor, the mutant motors display decreased velocities, run lengths, and landing rates. In this seminar, I will present these findings and discuss our newest structure-function and single-molecule optical tweezers studies that provide new insights into the coordination and force generation of the superprocessive KIF1A.   

Signaling and mechanics in mitotic spindle assembly

During mitosis a bipolar spindle structure is assembled in the cell to equally partition the duplicated genetic material into daughter cells. Faulty spindle assembly can cause genomic errors that kill the cell or promote cancer development. Proper spindle assembly relies on the coordinated activities of numerous molecules that play different roles, from structure and mechanics to signaling and quality surveillance. In this talk I will introduce our recent theoretical modeling in both the signaling and mechanical aspects of spindle assembly. For signaling, we modeled the intracellular spatiotemporal dynamics of the spindle assembly checkpoint proteins – molecules that survey the attachments between the chromosomes and the spindle. Our model proposes a novel mechanistic link between the observed spatial regulation of the checkpoint proteins and noise robustness of the checkpoint silencing process. The model provides mechanistic explanations for many intriguing mitotic phenomena, for instance, how the checkpoint mechanism facilitates bipolar spindle assembly in cancer cells with extra centrosomes. Furthermore, in the mechanical aspect, we modeled the mechanical dynamics that drives the movements of centrosomes in a mitotic cell and examined the resulting spindle geometry. Our model predicts the most critical biophysical factors that enable bipolar spindle formation in the presence of both normal and extra centrosome numbers.   

Adaptation and evolution in flagellar motors

The bacterial flagellar motor (BFM) is a membrane-embedded, ion-driven rotary nanomachine responsible for the motility of the majority of known bacterial species. Torque in the BFM is generated at the interface between the motor's rotor and transmembrane stator units, which are tethered to the peptidoglycan layer of the bacterial cell wall. Each stator unit contains an ion-binding site, allowing the motor to use the energy from the passage of ions (usually protons or sodium) down a transmembrane gradient for torque generation. Recent high-resolution structures of the BFM's stator units have provided considerable new insight into the possible mechanism of torque generation in the motor; these new structures also necessitate a re-examination of several predictions made by previous models. Here, we present a model for torque generation in the E. coli flagellar motor rooted in this new structural understanding. Further, using both structural information and experiments on motor dynamics from several bacterial species, we analyze how adaptation under various selective pressures has shaped the diversity of flagellar motors across bacterial lineages.