Biology subjects

Pedaci, F.

Publications and source records attributed to Pedaci, F..

3 recordsLinked to original sources

bioRxiv2018

Kinetic analysis methods applied to single motor protein trajectories

Molecular motors convert chemical or electrical energy into mechanical displacement, either linear or rotary. Under ideal circumstances, single-molecule measurements can spatially and temporally resolve individual steps of the motor, revealing important properties of the underlying mechanochemical process. Unfortunately, steps are often hard to resolve, as they are masked by thermal noise. In such cases, details of the mechanochemistry can nonetheless be recovered by analyzing the fluctuations in the recorded traces. Here, we expand upon existing statistical analysis methods, providing two new avenues to extract the motor step size, the effective number of rate-limiting chemical states per translocation step, and the compliance of the link between the motor position and the probe particle. We first demonstrate the power and limitations of these methods using simulated molecular motor trajectories, and we then apply these methods to experimental data of kinesin, the bacterial flagellar motor, and F1-ATPase.

biophysics↗

bioRxiv2017

A catch-bond drives stator mechanosensitivity in the Bacterial Flagellar Motor

The bacterial flagellar motor (BFM) is the rotary motor which powers the swimming and swarming of many motile bacteria. The torque is provided by stator units, ion motive force powered ion channels known to assemble and disassemble dynamically in the BFM. This turnover is mechano-sensitive, with the number of engaged units dependent upon the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechano-sensitivity is unknown. Here we directly measure the kinetics of arrival and departure of the stator units in individual wild-type motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. Obtaining the real-time stator stoichiometry before and after periods of forced motor stall, we measure both the number of active stator units at steady-state as a function of the load and the kinetic association and dissociation rates, by fitting the data to a reversible random sequential adsorption model. Our measurements indicate that BFM mechano-sensing relies on the dissociation rate of the stator units, which decreases with increasing load, while their association rate remains constant. This implies that the lifetime of an active stator unit assembled within the BFM increases when a higher force is applied to its anchoring point in the cell wall, providing strong evidence that a catch-bond mechanism can explain the mechano-sensitivity of the BFM.

biophysics↗

bioRxiv2017

Impact of fluorescent protein fusions on the bacterial flagellar motor

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects. Such insight, however, is still lacking for many applications of fluorescent fusions. This is particularly relevant in the study of the internal dynamics of motor protein complexes, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the bacterial flagellar motor (BFM) complex have previously been used to successfully unveil the internal subunit dynamics of the motor. Here we report the effects of three different fluorescent proteins fused to the stator, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry in the complex remained unaffected. MotB fusions decreased the rotation-direction switching frequency of single motors and induced a novel BFM behavior: a bias-dependent asymmetry in the speed attained in the two rotation directions. All these effects could be mitigated by the insertion of a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions on BFM dynamics and their alleviation--new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.\n\nAuthor summaryMuch of what is known about the biology of proteins was discovered by fusing them to fluorescent proteins that allow detection of their location. But the label comes at a cost: the presence of the tag can alter the behavior of the protein of interest in unforeseen, yet biologically relevant ways. These side effects limit the depth to which fluorescent proteins can be used to probe protein function. One of the systems that has been successfully studied with fluorescent fusions for which these effects have not been addressed are dynamic protein complexes that carry out mechanical work. We examined how fluorescent proteins fused to a component of the bacterial flagellar motor complex impacts its function. Our findings show that the fusion proteins altered biologically relevant dynamical properties of the motor, including induction of a novel mechanical behavior, and demonstrate an approach to alleviate this. These results advance our ability to dissect the bacterial flagellar motor, and the internal dynamics of protein complexes in general, with fluorescent fusion proteins while causing minimal perturbation.

biophysics↗