Direct determination of molar absorption coefficients of several molecules in the lowest excited singlet states.

Molar absorption coefficient of the lowest excited state is an indispensable information for the quantitative investigation of photochemical reactions by means of transient absorption spectroscopy. In the present work, we quantitatively estimated the molar absorption coefficients of the S1 state of the solute in three solution systems, Rhodamine B in ethanol, ZnTPP in DMF and N,N'-bis(2,6-diisopropylphenyl)terrylene-3,4,11,12-tetracarboxydiimide (TDI) in chloroform, by perfectly bleaching the ground state molecules using the picosecond 532-nm laser pulse with a large number of photons. These solution systems were selected because no obvious photodegradation was detected in the present range of the excitation intensity. The molar absorption coefficient obtained by this method was verified by the numerical analysis of the excitation intensity dependence of the transient absorbance by taking into account the inner filter effect (absorption of the excitation light by the S1 state produced by the leading part of the pump pulse) and the decrease of the ground state molecules by the pump process (depletion). In addition, these molar absorption coefficients were confirmed by the comparison of relations between the excitation intensity and the transient absorbance of the S1 state under the condition where the fraction of the excited solute is ≪ 10% by the femtosecond pulsed laser excitation. From these results, the error of the molar absorption coefficients was estimated to be < 5%. These values can be used as reference ones for the estimation of molar absorption coefficients of other systems leading to the quantitative elucidation of the photochemical reactions detected by the transient absorption spectroscopy.

Canine Orientia tsutsugamushi infection: report of a case and its epidemicity.

A lethargic household dog was referred to a private hospital in Japan. Diagnosis was carried out by the polymerase chain reaction (PCR) method developed for human Orientia tsutsugamushi infection using the dog's anticoagulated peripheral blood. Karp, Kato and Kuroki-type genomes were detected and the dog was diagnosed with O. tsutsugamushi infection. These findings demonstrate that dogs can act as a host for O. tsutsugamushi and the PCR method developed for human beings can be used for the diagnosis of canine O. tsutsugamushi infection. A concurrent epidemiological study examined 10 asymptomatic dogs that were fed in the same area as the sick dog. Kuroki-type genome in all dogs, Gilliam-type genome in 6 dogs and Kawasaki-type genome in 3 dogs were detected. These results provide further evidence that dogs can be naturally infected with O. tsutsugamushi outdoors and that dogs play a role as a host in the lifecycle of O. tsutsugamushi.

Machine learning based prediction of lattice thermal conductivity for half-Heusler compounds using atomic information.

Half-Heusler compound has drawn attention in a variety of fields as a candidate material for thermoelectric energy conversion and spintronics technology. When the half-Heusler compound is incorporated into the device, the control of high lattice thermal conductivity owing to high crystal symmetry is a challenge for the thermal manager of the device. The calculation for the prediction of lattice thermal conductivity is an important physical parameter for controlling the thermal management of the device. We examined whether lattice thermal conductivity prediction by machine learning was possible on the basis of only the atomic information of constituent elements for thermal conductivity calculated by the density functional theory in various half-Heusler compounds. Consequently, we constructed a machine learning model, which can predict the lattice thermal conductivity with high accuracy from the information of only atomic radius and atomic mass of each site in the half-Heusler type crystal structure. Applying our results, the lattice thermal conductivity for an unknown half-Heusler compound can be immediately predicted. In the future, low-cost and short-time development of new functional materials can be realized, leading to breakthroughs in the search of novel functional materials.

Dynamics of group II chaperonin and prefoldin probed by 13C NMR spectroscopy.

Group II chaperonin (CPN) cooperates with prefoldin (PFD), which forms a jellyfish-shaped heterohexameric complex with a molecular mass of 87 kDa. PFD captures an unfolded protein with the tentacles and transfers it to the cavity of CPN. Although X-ray crystal structures of CPN and PFD have been reported, no structural information has been so far available for the terminal regions of the PFD tentacles nor for the C-terminal segments of CPNs, which were regarded to be functionally significant in the previous studies. Here we report 13C NMR analyses on archaeal PFD, CPN, and their complex, focusing on those structurally uncharacterized regions. The PFD and CPN complexes selectively labeled with 13C at methionyl carbonyl carbons were separately and jointly subjected to NMR measurements. 13C NMR spectral data demonstrated that the N-terminal segment of the alpha and beta subunits of PFD as well as the C-terminal segments of the CPN hexadecamer retain significant degrees of freedom in internal motion even in the complex with a molecular mass of 1.1 MDa.

Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate.

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. The manner by which prefoldin interacts with a group II chaperonin is poorly understood. Here, we have examined the prefoldin interaction site in the archaeal group II chaperonin, comparing the interaction of two Thermococcus chaperonins and their mutants with Pyrococcus prefoldin by surface plasmon resonance. We show that the mutations of Lys250 and Lys256 of Thermococcus alpha chaperonin residues to Glu residues increase the affinity to Pyrococcus prefoldin to the level of Thermococcus beta chaperonin and Pyrococcus chaperonin, indicating that their Glu250 and Glu256 residues of the helical protrusion region are responsible for relatively stronger binding to Pyrococcus prefoldin than Thermococcus alpha chaperonin. Since the putative chaperonin binding sites in the distal ends of Pyrococcus prefoldin are rich in basic residues, electrostatic interaction seems to be important for their interaction. The substrate protein transfer rate from prefoldin correlates well with its affinity for chaperonin.

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1.

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two alpha subunits and four beta subunits with the structure of a double beta-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (alpha1, alpha2, beta1, and beta2) from T. KS-1. All of them (alpha1-beta1, alpha2-beta1, alpha1-beta2, and alpha2-beta2) exist as alpha(2)beta(4) heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the beta1 subunit interacted with the chaperonins more strongly than those with the beta2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.

Structure and molecular dynamics simulation of archaeal prefoldin: the molecular mechanism for binding and recognition of nonnative substrate proteins.

Prefoldin (PFD) is a heterohexameric molecular chaperone complex in the eukaryotic cytosol and archaea with a jellyfish-like structure containing six long coiled-coil tentacles. PFDs capture protein folding intermediates or unfolded polypeptides and transfer them to group II chaperonins for facilitated folding. Although detailed studies on the mechanisms for interaction with unfolded proteins or cooperation with chaperonins of archaeal PFD have been performed, it is still unclear how PFD captures the unfolded protein. In this study, we determined the X-ray structure of Pyrococcus horikoshii OT3 PFD (PhPFD) at 3.0 A resolution and examined the molecular mechanism for binding and recognition of nonnative substrate proteins by molecular dynamics (MD) simulation and mutation analyses. PhPFD has a jellyfish-like structure with six long coiled-coil tentacles and a large central cavity. Each subunit has a hydrophobic groove at the distal region where an unfolded substrate protein is bound. During MD simulation at 330 K, each coiled coil was highly flexible, enabling it to widen its central cavity and capture various nonnative proteins. Docking MD simulation of PhPFD with unfolded insulin showed that the beta subunit is essentially involved in substrate binding and that the alpha subunit modulates the shape and width of the central cavity. Analyses of mutant PhPFDs with amino acid replacement of the hydrophobic residues of the beta subunit in the hydrophobic groove have shown that beta Ile107 has a critical role in forming the hydrophobic groove.