Temperature Dependence of the Rotation and Hydrolysis Activities of F1-ATPase

F1-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 F1-ATPase over the temperature range 4–50°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 × 106 M−1 s−1 at 4°C to 4.3 × 107 M−1 s−1 at 40°C, whereas the torque estimated at 2 mM ATP remained around 40 pN·nm over 4–50°C. The rotation was stepwise at 4°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°C. F1-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°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

Inverse regulation of rotation of F1-ATPase by the mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase.

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A programmable optical angle clamp for rotary molecular motors

Optical tweezers are widely used for experimental investigation of linear molecular motors. The rates and force dependence of steps in the mechanochemical cycle of linear motors have been probed giving detailed insight into motor mechanisms. With similar goals in mind for rotary molecular motors we present here an optical trapping system designed as an angle clamp to study the bacterial flagellar motor and F(1)-ATPase. The trap position was controlled by a digital signal processing board and a host computer via acousto-optic deflectors, the motor position via a three-dimensional piezoelectric stage and the motor angle using a pair of polystyrene beads as a handle for the optical trap. Bead-pair angles were detected using back focal plane interferometry with a resolution of up to 1 degrees , and controlled using a feedback algorithm with a precision of up to 2 degrees and a bandwidth of up to 1.6 kHz. Details of the optical trap, algorithm, and alignment procedures are given. Preliminary data showing angular control of F(1)-ATPase and angular and speed control of the bacterial flagellar motor are presented

Resolving stepping rotation in Thermus thermophilus H+-ATPase/synthase with an essentially drag-free probe

Vacuole-type ATPases (VoV1) and FoF1 ATP synthases couple ATP hydrolysis/synthesis in the soluble V1 or F1 portion with proton (or Na+) flow in the membrane-embedded Vo or Fo portion through rotation of one common shaft. Here we show at submillisecond resolutions the ATP-driven rotation of isolated V1 and the whole VoV1 from Thermus thermophilus, by attaching a 40-nm gold bead for which viscous drag is almost negligible. V1 made 120° steps, commensurate with the presence of three catalytic sites. Dwells between the steps involved at least two events other than ATP binding, one likely to be ATP hydrolysis. VoV1 exhibited 12 dwell positions per revolution, consistent with the 12-fold symmetry of the Vo rotor in T. thermophilus. Unlike F1 that undergoes 80°–40° substepping, chemo-mechanical checkpoints in isolated V1 are all at the ATP-waiting position, and Vo adds further bumps through stator–rotor interactions outside and remote from V1

Viral nanomotors for packaging of dsDNA and dsRNA

While capsid proteins are assembled around single-stranded genomic DNA or RNA in rod-shaped viruses, the lengthy double-stranded genome of other viruses is packaged forcefully within a preformed protein shell. This entropically unfavourable DNA or RNA packaging is accomplished by an ATP-driven viral nanomotor, which is mainly composed of two components, the oligomerized channel and the packaging enzymes. This intriguing DNA or RNA packaging process has provoked interest among virologists, bacteriologists, biochemists, biophysicists, chemists, structural biologists and computational scientists alike, especially those interested in nanotechnology, nanomedicine, AAA+ family proteins, energy conversion, cell membrane transport, DNA or RNA replication and antiviral therapy. This review mainly focuses on the motors of double-stranded DNA viruses, but double-stranded RNA viral motors are also discussed due to interesting similarities. The novel and ingenious configuration of these nanomotors has inspired the development of biomimetics for nanodevices. Advances in structural and functional studies have increased our understanding of the molecular basis of biological movement to the point where we can begin thinking about possible applications of the viral DNA packaging motor in nanotechnology and medical applications

Raman spectroscopy as a tool to determine the thermal maturity of organic matter : application to sedimentary, metamorphic and structural geology

Raman spectrometry is a rapid, non-destructive alternative to conventional tools employed to assess the thermal alteration of organic matter (OM). Raman may be used to determine vitrinite reflectance equivalent OM maturity values for petroleum exploration, to provide temperature data for metamorphic studies, and to determine the maximum temperatures reached in fault zones. To achieve the wider utilisation of Raman, the spectrum processing method, and the positions and nomenclature of Raman bands and parameters, all need to be standardized. We assess the most widely used Raman parameters as well as the best analytical practices that have been proposed. Raman band separation and G-band full-width at half-maximum are the best parameters to estimate the maturity for rocks following diagenesis–metagenesis. For metamorphic studies, the ratios of band areas after performing deconvolution are generally used. Further work is needed on the second-order region, as well as assessing the potential of using integrated areas on the whole spectrum, to increase the calibrated temperature range of Raman parameters. Applying Raman spectroscopy on faults has potential to be able to infer both temperature and deformation processes. We propose a unified terminology for OM Raman bands and parameters that should be adopted in the future. The popular method of fitting several functions to a spectrum is generally unnecessary, as Raman parameters determined from an un-deconvoluted spectrum can track the maturity of OM. To progress the Raman application as a geothermometer a standardized approach must be developed and tested by means of an interlaboratory calibration exercise using reference materials

Carotenoid Nostoxanthin Production by Sphingomonas sp. SG73 Isolated from Deep Sea Sediment

http://www.godac.jamstec.go.jp/darwin/cruise/natsushima/nt99-13/ehttp://www.godac.jamstec.go.jp/darwin/cruise/shinsei_maru/ks-18-j02/ehttp://www.godac.jamstec.go.jp/darwin/cruise/yokosuka/yk18-04/ehttp://www.godac.jamstec.go.jp/darwin/cruise/kaimei/km19-02/