Temperature dependence of the rotation and hydrolysis activities of F1-ATPase.

F(1)-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F(1)-ATPase over the temperature range 4-50 degrees C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 x 10(6) M(-1) s(-1) at 4 degrees C to 4.3 x 10(7) M(-1) s(-1) at 40 degrees C, whereas the torque estimated at 2 mM ATP remained around 40 pN.nm over 4-50 degrees C. The rotation was stepwise at 4 degrees C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65 degrees C. F(1)-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30 degrees C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.

Towards single biomolecule handling and characterization by MEMS.

Applications of microelectromechanical systems (MEMS) technology are widespread in both industrial and research fields providing miniaturized smart tools. In this review, we focus on MEMS applications aiming at manipulations and characterization of biomaterials at the single molecule level. Four topics are discussed in detail to show the advantages and impact of MEMS tools for biomolecular manipulations. They include the microthermodevice for rapid temperature alternation in real-time microscopic observation, a microchannel with microelectrodes for isolating and immobilizing a DNA molecule, and microtweezers to manipulate a bundle of DNA molecules directly for analyzing its conductivity. The feasibilities of each device have been shown by conducting specific biological experiments. Therefore, the development of MEMS devices for single molecule analysis holds promise to overcome the disadvantages of the conventional technique for biological experiments and acts as a powerful strategy in molecular biology.

Single DNA molecule isolation and trapping in a microfluidic device.

This paper presents a systematic method to isolate and trap long single DNA segments between integrated electrodes in a microfluidic environment. Double stranded lambda-DNA molecules are introduced in a microchip and are isolated by electrophoretic force through microfluidic channels. Downstream, each individual molecule is extended and oriented by ac dielectrophoresis (900 kHz, 1 MV m(-1)) and anchored between aluminium electrodes. With a proper design, a long DNA segment (up to 10 microm) can be instantly captured in stretched conformation, opening way for further assays.

One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar.

F(1)-ATPase is a rotary molecular motor in which the central gamma-subunit rotates inside a cylinder made of alpha(3)beta(3)-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the beta-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F(1) rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F(1) molecules that carried a bead duplex on the gamma-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 x 10(7) M(-1)s(-1). A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F(1) produced the same torque of approximately 40 pN.nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.