Schematic views of the buffer exchange chamber for single biomotor observation. Inlet and outlet micro-one-way valves are placed on the tip of a tube connected to the coverslip of the chamber. While the valves are opened with forward pressure, the exchange buffer is injected into the chamber (upper illustration). After replacing the buffer completely, the valves are closed with backward pressure (lower illustration).
(a) The buffer exchange chamber on an observation stage of the microscope. (b) SEM image of the valve. (c) Photos of the parylene micro-one-way valve. The valve is moved upside when opened (left) and becomes flat when closed (right).
Flow characteristics with (blue circles) and without (white circles) the parylene valve.
inhibition of -ATPase. (a) Scheme of inhibition of -ATPase. is considered to stabilize ADP inhibition state, in which the -ATPase fails to dissociate ADP from the catalytic site (Ref. 8). (b) Time course of inhibition in the fabricated chamber. Here, we observed -ATPase at 2 mM ATP. After 45 s, valves are opened and containing buffers are introduced into the chamber. We close the valves at 51 s and continue recording. Arrows indicate duration of the actively rotating -ATPases after closing the valves. (c) Duration of the active -ATPase in containing ATP buffer. Solid line shows a single-exponential function with (rate of conversion from the rotation to the pause) .
Stator-rotor cross-linking experiment under fabricated chamber. (a) Scheme of disulfide bond formation between rotor and stator . The disulfide bond is reduced by DTT and cross-linked by oxidation with DTNB. (b) Time course of rotation of mutant -ATPase. -ATPase rotates at 200 nM ATP under DTT-containing buffer [(i) phase] and then DTNB-containing buffer is introduced into the chamber (open valve first time). Rotation was paused after cross-linking by disulfide bond formation between the rotor and the stator [(ii) phase] and then DTT-containing buffer was introduced (open valve second time).
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