Nonequilibrium energetics of single molecule translational motor kinesin was investigated by measuring heat dissipation from the violation of the fluctuation-response relation of a probe attached to the motor using optical tweezers. The sum of the dissipation and work did not amount to the input free energy change, indicating large hidden dissipation exists. Possible sources of the hidden dissipation were explored by analyzing the Langevin dynamics of the probe, which incorporates the two-state Markov stepper as a kinesin model. We conclude that internal dissipation is dominant.Kinesin-1 (hereafter called kinesin) is a molecular motor that transports various cargos along microtubules throughout the cell [1,2]. Single molecule kinesin takes 8 nm steps [3] per ATP hydrolysis [4,5] on a microtubule rail and generates ≈7 pN maximum force [6][7][8]. The two catalytic sites (heads) hydrolyze ATP in a "hand-over-hand" manner that mimics bipedal walking [9-11] by alternating its two heads in coordination with different nucleotide/microtubule binding states [12]. Kinesin shows backward steps occasionally at no load and frequently at high loads [13][14][15]. Recent experiments indicate that the biased unidirectional motion is achieved by regulating selective binding/unbinding of the head to/from the appropriate binding site [16][17][18][19][20]. Contrary to the molecular mechanism of the motility, the thermodynamic energetics of the motor is poorly understood due to kinesin's stochastic and nonequilibrium behavior.The energetics of single-molecule motors were historically discussed when their stall forces were measured [21]. Kinesin's stall force of ≈7 pN indicates that maximum work per 8-nm step (≈56 pN•nm) is smaller than the physiological free energy change per ATP hydrolysis (≈85 pN•nm). This is in contrast to rotary motor F 1 -ATPase, whose stall force explains all input free energy [22]. Kinesin's inefficient work at stall has been regarded as an "open problem" [23]. However, it may not be appropriate to evaluate kinesin efficiency from the stall force for two reasons. First, it is believed that kinesin consumes ATP at backsteps instead of synthesizing ATP [13][14][15], indicating that the stall condition is not thermodynamically (quasi-)static. Second, the physiological role of kinesin is to carry vesicles against viscous media, meaning that the input energy is dissipated as "heat" rather than "work." Thus, measuring the "dissipation" from the motor is essential when discussing kinesin's nonequilibrium energetics in physiologically relevant conditions.The Harada-Sasa equality is best suited for this purpose [24,25] :Here, J x is total heat dissipation per unit time from the system through specific degrees of freedom indicated with subscript x. γ is viscous drag, and x  v is mean velocity, where  denotes the ensemble average. () Cf , with frequency f, is a Fourier transform of the correlation function of velocity fluctuations,is a Fourier transform of the velocity response function, and the prime indicates the re...

Feedback-tracking microrheology, developed to study nonequilibrium mechanics of active materials, revealed glassy cell dynamics.

F1-ATPase, the catalytic part of FoF1-ATP synthase, rotates the central gamma subunit within the alpha3beta3 cylinder in 120 degrees steps, each step consuming a single ATP molecule. However, how the catalytic activity of each beta subunit is coordinated with the other two beta subunits to drive rotation remains unknown. Here we show that hybrid F1 containing one or two mutant beta subunits with altered catalytic kinetics rotates in an asymmetric stepwise fashion. Analysis of the rotations reveals that for any given beta subunit, the subunit binds ATP at 0 degrees, cleaves ATP at approximately 200 degrees and carries out a third catalytic event at approximately 320 degrees. This demonstrates the concerted nature of the F1 complex activity, where all three beta subunits participate to drive each 120 degrees rotation of the gamma subunit with a 120 degrees phase difference, a process we describe as a 'sequential three-site mechanism'.

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