Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering

To monitor the fast compaction process during protein folding, we have used a stopped-flow small-angle X-ray scattering technique combined with a two-dimensional charge-coupled device-based X-ray detector that makes it possible to improve the signal-to-noise ratio of data dramatically, and measured the kinetic refolding reaction of alpha-lactalbumin. The results clearly show that the radius of gyration and the overall shape of the kinetic folding intermediate of alpha-lactalbumin are the same as those of the molten globule state observed at equilibrium. Thus, the identity between the kinetic folding intermediate and the equilibrium molten globule state is firmly established. The present results also suggest that the folding intermediate is more hydrated than the native state and that the hydrated water molecules are dehydrated when specific side-chain packing is formed during the change from the molten globule to the native state.     

Personality research with non-human primates: theoretical formulation and methods

Since the earliest periods of primatology study, researchers have been aware of animals’ consistent individual differences in behavior or personality. Many papers have been published on this subject, but they lacked a common theoretical and methodological background. The present work is an attempt to provide such theoretical and methodological foundations to this field of biological science. In the theoretical formulation section, “biological study of personality” is first derived as an extension of the ethology paradigm, and non-human primate personality research is subsequently characterized as its strategic component. In the methodology section, brief reviews and discussions are presented on subjective and objective personality assessment methods with non-human primates with a reference to the reliability and validity concepts in human psychometrics. The work provides a theoretical framework and methodological suggestions for non-human primate personality research.     

Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.     

Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8

FtsH is a cytoplasmic membrane-integrated, ATP-dependent metalloprotease, which processively degrades both cytoplasmic and membrane proteins in concert with unfolding. The FtsH protein is divided into the N-terminal transmembrane region and the larger C-terminal cytoplasmic region, which consists of an ATPase domain and a protease domain. We have determined the crystal structures of the Thermus thermophilus FtsH ATPase domain in the nucleotide-free and AMP-PNP- and ADP-bound states, in addition to the domain with the extra preceding segment. Combined with the mapping of the putative substrate binding region, these structures suggest that FtsH internally forms a hexameric ring structure, in which ATP binding could cause a conformational change to facilitate transport of substrates into the protease domain through the central pore.     

Regeneration processes of a boreal forest in Kamchatka with special reference to the contribution of sprouting to population maintenance

We studied regeneration patterns of three tree species Picea ajanensis, Betula platyphylla and Populus tremula from 1998 to 2000 in the Central Depression of the Kamchatka Peninsula. We paid special attention to the contribution of sprouting to their regeneration. P. ajanensis was the only species that regenerated by seedling. In a 40 × 40 m study plot, the density of P. ajanensis saplings < 2.0 cm in diameter at basal area (DBH) was 1132, and this was the highest among the three species studied. The number of saplings 2 cm in DBH declined sharply with size class. The spatial distribution of P. ajanensis saplings (< 2 cm in DBH) showed a significant positive correlation with that of adult trees and a negative correlation with that of gaps. These trends were not changed after re-measurement in 2000, although nearly half of the juveniles had died or been injured during the two years. These results suggest that small Picea saplings prefer habitats under the canopy of adult trees rather than in gaps for establishment. Most small individuals of B. platyphylla were produced from sprouts. The number of saplings in the smallest size class (< 2 cm in DBH) was much less than that of P. ajanensis, although the number of larger individuals did not decrease remarkably. The spatial distribution of B. platyphylla saplings showed a positive correlation with that of adult trunks and a negative correlation with that of canopy trees of P. ajanensis. These results suggest an effective contribution of sprouts to the regeneration of B. platyphylla. P. tremula was the only species that could invade big gaps and produce many root suckers efficiently. There were 181 suckers of P. tremula in the smallest size class (< 2 cm in DBH) in the study plot, although the number of saplings 2 cm in DBH declined abruptly. The spatial distribution of saplings of this species showed a slight positive correlation with that of gaps, and negative correlation with that of adult trees of B. platyphylla, P. ajanensis, and P. tremula. The root suckering strategy of P. tremula might be adaptive under severe conditions in high-latitude regions. Our data suggest, however, that it does not necessarily contribute to regeneration in mature forests. The three component species in this forest did not seem to utilize canopy gaps for regeneration; we suggest that gap dynamics do not work in this forest. The sparse canopy, which is a typical character of forests in high-latitude regions, might be a consequence of high mortalities of seedlings and root suckers inside gaps.     

Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on chlamydomonas

An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene. AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type. By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3. The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1666 amino acid residues. This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na(+)/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure. Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake. These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake. Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type.     

NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif

Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.