42 research outputs found
Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format
BACKGROUND: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. RESULTS: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. CONCLUSIONS: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome
Upgrading of shielding for rare decay search in CANDLES
In the CANDLES experiment aiming to search for the very rare neutrino-less double beta decays (0νββ) using 48Ca, we introduced a new shielding system for high energy γ-rays from neutron captures in massive materials near the detector, in addition to the background reduction for 232Th decays in the 0νββ target of CaF2 crystals. The method of background reduction and the performance of newly installed shielding system are described
Cost-Effective Method for Free-Energy Minimization in Complex Systems with Elaborated Ab Initio Potentials
We
describe a method to locate stationary points in the free-energy
hypersurface of complex molecular systems using high-level correlated <i>ab initio</i> potentials. In this work, we assume a combined
QM/MM description of the system although generalization to full <i>ab initio</i> potentials or other theoretical schemes is straightforward.
The free-energy gradient (FEG) is obtained as the mean force acting
on relevant nuclei using a dual level strategy. First, a statistical
simulation is carried out using an appropriate, low-level quantum
mechanical force-field. Free-energy perturbation (FEP) theory is then
used to obtain the free-energy derivatives for the target, high-level
quantum mechanical force-field. We show that this composite FEG-FEP
approach is able to reproduce the results of a standard free-energy
minimization procedure with high accuracy, while simultaneously allowing
for a drastic reduction of both computational and wall-clock time.
The method has been applied to study the structure of the water molecule
in liquid water at the QCISD/aug-cc-pVTZ level of theory, using the
sampling from QM/MM molecular dynamics simulations at the B3LYP/6-311+G(d,p)
level. The obtained values for the geometrical parameters and for
the dipole moment of the water molecule are within the experimental
error, and they also display an excellent agreement when compared
to other theoretical estimations. The developed methodology represents
therefore an important step toward the accurate determination of the
mechanism, kinetics, and thermodynamic properties of processes in
solution, in enzymes, and in other disordered chemical systems using
state-of-the-art <i>ab initio</i> potentials