Optical tweezers have enabled single-molecule measurements of kinesin mechanics and energetics but face challenges for in vivo applications due to photothermal effects and refractive-index-dependent force calibration. Here, we developed electromagnetic tweezers featuring horizontal magnetic field geometry that enables force application along the microtubule axis for translational motor measurements. We validated the platform by reproducing established kinesin mechanics: stall force of ~6 pN and, using the Harada-Sasa equality to quantify nonequilibrium dissipation, an estimated energy conversion efficiency of ~20%, both consistent with optical tweezers results. We further demonstrated that FDT violations increase with ATP concentration following Michaelis-Menten kinetics, consistent with kinesin's enzymatic turnover. These results establish electromagnetic tweezers as a complementary tool offering advantages in high-throughput measurements and environment-independent force calibration, providing a foundation for future applications where these capabilities are essential.

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Cells robustly maintain metabolic functions despite environmental fluctuations that broadly alter reaction kinetics. However, kinetic models of cellular metabolism often exhibit fragility, losing stability with minor parameter changes. This discrepancy suggests that cells possess metabolic regulations that preserve the stable state under environmental perturbations. To understand the principles of metabolic robustness, we investigated the effects of temperature changes on a kinetic model of Escherichia coli central metabolism. We found that a gradual temperature decrease destabilized the metabolic state, leading to an abrupt shift to a new state in which glycolytic and tricarboxylic acid cycle (TCA cycle) fluxes decreased, and ATP production efficiency dropped. This shift was triggered by an elevated ATP/ADP ratio, which created a bottleneck in the glycolytic pathway. To assess the relationship between the destabilization and the ATP/ADP ratio, we introduced a rapid ATP–ADP exchange reaction and prevented this surge in the ATP/ADP ratio. Under the ATP/ADP ratio homeostasis, ATP production efficiency remained high across temperatures. Furthermore, we demonstrated that the destabilization was also avoided by altering enzyme abundances through sampling multiple stable steady states under cold conditions. The predicted enzyme regulation to maintain high ATP production efficiency was consistent with experimental observations of E. coli at low temperatures. Our findings indicate that balancing key cofactors, particularly the ATP/ADP ratio, is crucial for preserving metabolic stability under environmental perturbations.

Metabolic dynamics simulated using a kinetic model often exhibit fragility to small parameter perturbations. To elucidate the mechanism underlying this fragility, we analyzed how the metabolic state changes in response to temperature shift. As a result, we found that a gradual decrease in temperature destabilized the metabolic state, leading to an abrupt transition to a state with reduced glycolytic flux and ATP yield, and increased ATP/ADP ratio (right panels). Furthermore, introducing a hypothetical reaction that maintains a constant ATP/ADP ratio suppressed this transition. These findings highlight the importance of maintaining cofactor balance for the stability of the metabolic system.

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We previously demonstrated that sub-terahertz irradiation significantly accelerates the formation of hydration structures in protein aqueous solutions under nonequilibrium conditions, immediately after mixing. To monitor this phenomenon, we developed a microwave dielectric measurement technique capable of sensitively detecting time-dependent changes in hydration under sub-terahertz irradiation. This method utilizes the dielectric-dependent modulation of multiply reflected signals in short-path-length samples (Sugiyama et al., Nat. Commun. 14:2825, 2023). However, the physical origin of the observed signal remained unclear, limiting its broader applicability. In this study, we identify the origin as destructive interference between reflections at the probe–sample interface and the bottom surface of the sample container, which arises uniquely under a short-path-length condition satisfying d=λ/4. This finding establishes a clear measurement principle and enables direct evaluation of dielectric changes in biomolecular hydration from raw reflection data without converting to complex permittivity.

We present a simple and sensitive microwave reflection method that exploits destructive interference occurring when the sample path length equals one-quarter of the effective wavelength (d = λ/4). Under this condition, reflections from the probe–sample interface and the sample-container interface are in antiphase, producing a pronounced return-loss peak. The peak frequency shifts with changes in static dielectric permittivity, enabling direct evaluation of changes in biomolecular hydration from raw reflection spectra without converting to complex permittivity.

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