A reader has drawn our attention to a mistake in our manuscript. Eqn (7) (Formula Presented). Should have read (Formula Presented) We have also corrected different mistakes in the supplementary information file, in the equations in section 1. A corrected supplementary information file is provided. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.

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 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.

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, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation-new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.