Kumiko Hayashi

Improving Signal/Noise Resolution in Single-Molecule Experiments Using Molecular Constructs with Short Handles

We investigate unfolding/folding force kinetics in DNA hairpins exhibiting two and three states with newly designed short dsDNA handles (29 bp) using optical tweezers. We show how the higher stiffness of the molecular setup moderately enhances the signal/noise ratio (SNR) in hopping experiments as compared to conventional long-handled constructs (≅700 bp). The shorter construct results in a signal of higher SNR and slower folding/unfolding kinetics, thereby facilitating the detection of otherwise fast structural transitions. A novel analysis, as far as we are aware, of the elastic properties of the molecular setup, based on high-bandwidth measurements of force fluctuations along the folded branch, reveals that the highest SNR that can be achieved with short handles is potentially limited by the marked reduction of the effective persistence length and stretch modulus of the short linker complex.

Catalysis-Enhancement via Rotary Fluctuation of F1-ATPase

Protein conformational fluctuations modulate the catalytic powers of enzymes. The frequency of conformational fluctuations may modulate the catalytic rate at individual reaction steps. In this study, we modulated the rotary fluctuation frequency of F1-ATPase (F1) by attaching probes with different viscous drag coefficients at the rotary shaft of F1. Individual rotation pauses of F1 between rotary steps correspond to the waiting state of a certain elementary reaction step of ATP hydrolysis. This allows us to investigate the impact of the frequency modulation of the rotary fluctuation on the rate of the individual reaction steps by measuring the duration of rotation pauses. Although phosphate release was significantly decelerated, the ATP-binding and hydrolysis steps were less sensitive or insensitive to the viscous drag coefficient of the probe. Brownian dynamics simulation based on a model similar to the Sumi-Marcus theory reproduced the experimental results, providing a theoretical framework for the role of rotational fluctuation in F1 rate enhancement.

Viscosity and drag force involved in organelle transport: Investigation of the fluctuation dissipation theorem

We observed the motion of an organelle transported by motor proteins in cells using fluorescence microscopy. Particularly, among organelles, the mitochondria in PC12 cells were studied. A mitochondrion was dragged at a constant speed for several seconds without pausing. We investigated the fluctuation dissipation theorem for this constant drag motion by comparing it with the motion of Brownian beads that were incorporated into the cells by an electroporation method. We estimated the viscosity value inside cells from the diffusion coefficients of the beads. Then the viscosity value obtained by using the beads was found to be slightly lower than that obtained from the diffusion coefficient for the organelle motion via the Einstein relation. This discrepancy indicates the violation of the Einstein relation for the organelle motion.Graphical abstract

F-subunit reinforces torque generation in V-ATPase.

Vacuolar-type H+-pumping ATPases (V-ATPases) perform remarkably diverse functions in eukaryotic organisms. They are present in the membranes of many organelles and regulate the pH of several intracellular compartments. A family of V-ATPases is also present in the plasma membranes of some bacteria. Such V-ATPases function as ATP-synthases. Each V-ATPase is composed of a water-soluble domain (V1) and a membrane-embedded domain (Vo). The ATP-driven rotary unit, V

Measurements of the driving forces of bio-motors using the fluctuation theorem

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Giant enhancement of fluctuation in small biological systems under external fields

1, is composed of A, B, D, and F subunits. The rotary shaft (the DF subcomplex) rotates in the central cavity of the A3B3-ring (the catalytic hexamer ring). The D-subunit, which has a coiled-coil domain, penetrates into the ring, while the F-subunit is a globular-shaped domain protruding from the ring. The minimal ATP-driven rotary unit of V

Structural Fluctuation and Catalytic Function of F1-ATPase

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