Protein structural change upon ligand binding correlates with enzymatic reaction mechanism

Overall structural changes of enzymes in response to ligand binding were investigated by database analysis of 62 non-redundant enzymes whose ligand-unbound and ligand-bound forms were available in the Protein Data Bank. The results of analysis indicate that transferases often undergo large rigid-body domain motions upon ligand binding, while other enzymes, most typically, hydrolases, change their structures to a small extent. It was also found that the solvent accessibility of the substrate molecule was low in transferases but high in hydrolases. These differences are explained by the enzymatic reaction mechanisms. The transferase reaction requires the catalytic groups to be insulated from the water environment, and thus transferases bury the ligand molecule inside the protein by closing the cleft. On the other hand, the hydrolase reaction involves the surrounding water molecules and occurs at the protein surface, requiring only a small structural change.     

Hagfish (cyclostomata, vertebrata): searching for the ancestral developmental plan of vertebrates

The phylogenetic position of the hagfish remains enigmatic. In contrast to molecular data that suggest monophyly of the cyclostomes, several morphological features imply a more ancestral state of this animal compared with the lampreys. To resolve this question requires an understanding of the embryology of the hagfish, especially of the neural crest. The early development of the hagfish has long remained a mystery. We collected a shallow-water-dwelling hagfish, Eptatretus burgeri, set up an aquarium tank designed to resemble its habitat, and successfully obtained several embryos. By observing the histology and expression of genes known to play fundamental roles in the neural crest, we found that the hagfish crest develops as delaminating migratory cells, as in other vertebrates. We conclude that the delaminating neural crest is a vertebrate synapomorphy that seems to have appeared from the beginning of their evolutionary history, before the splitting away of the hagfish lineage.     

A parallel affinity purification method for selective isolation of polyubiquitinated proteins

We developed a parallel affinity purification (PAP) procedure, in which ubiquitinated proteins are purified from the cells that coexpress two affinity-tagged ubiquitins by sequential use of affinity chromatography specific to each tag. In contrast with previous procedures using a single affinity-tagged ubiquitin, the PAP eliminates highly abundant ubiquitin monomers and monoubiquitinated proteins to selectively enrich proteins bearing both affinity-tags, or poly- and multiubiquitinated proteins. Accordingly, it would serve as a powerful method to facilitate mass-spectrometric identification of ubiquitinated proteins.     

Alteration of apoptotic protease-activating factor-1 (APAF-1)-dependent apoptotic pathway during development of rat brain and liver.

Brain and liver extracts of rats at different stages after birth were examined for cytochrome c/dATP-dependent caspase (DEVDase)-activation (mitochondria pathway) in vitro. The caspase-activating activity in the brain extracts rapidly decreased after birth, reaching approximately 50 and 5%, at 1 and 2 weeks, respectively, of that in a 3-days- newborn sample, and essentially no caspase-activation was detected in the adult rat brain extracts. Such a dramatic change was not detected in the liver samples, suggesting that the observed abrogation of the cytochrome c-dependent mitochondria pathway after birth is a brain-specific event. In order to determine the factor(s) lacking in adult brain, we separately measured Apaf-1, procaspase 9, and pro-DEVDase activities using a supplementation assay. In adult brain, Apaf-1 activity was scarcely detected, while the tissue retained low but significant amounts of procaspase 9 (16% of that in the fetal tissue) and a pro-DEVDase (3.4%). In contrast, adult liver extracts retained relatively high levels of all of these factors. Immunoblot analyses clearly indicated that the expression of Apaf-1 and procaspase 3 is markedly suppressed within 4 weeks after birth in brain tissue while they are even expressed in adult liver. Considering these results together, we propose that, in the brain, the cytochrome c-dependent mitochondria pathway, which is essential for the programmed cell death during normal morphogenesis, is abrogated within 2-4 weeks after birth, whereas the pathway is still active in other adult tissues such as liver.     

Genetic studies on reference strains of mutans streptococci

Twenty four reference strains (serotype a-h) belonging to the mutans group of streptococci were compared for DNA fragment patterns of rDNA after treatment with Hind III. It was shown that Streptococcus cricetus (serotype a), S. rattus (serotype b), and S. downei (serotype h) reveals comparatively homogeneous patterns while S. mutans (serotype c, e and f) exhibits differences between the different serotypes as well as within single serotypes. S. sobrinus had an intermediary diversity. These data support the previous findings that S. mutans is heterogeneous at the serological, biochemical and genetical level.     

Phylogeny of the Eurasian freshwater turtles of the genus Mauremys Gray 1869 (Testudines), with special reference to a close affinity of Mauremys japonica with Chinemys reevesii

Phylogenetic relationships of the freshwater turtles of the genus Mauremys and representatives of several other batagurid genera were inferred from variations in 863 base positions of mitochondrial 12S and 16S rRNA genes. Results strongly suggested the non-monophyly of Mauremys by indicating the closest affinity of Mauremys japonica with Chinemys reevesii , the type species of the genus Chinemys . Recent morphological analyses of the batagurid genera suggested that Mauremys is a basal stock of the subfamily Geoemydinae, whereas Chinemys is a member of the subfamily Batagurinae as supported by several putative synapomorphs with other batagurine genera. It is thus probable that the morphological character states used to define Mauremys actually represent symplesiomorphy, and that morphological character states shared between Chinemys and other batagurine genera have resulted from convergence. Also, our results did not support a sister-group relationship between Mauremys annamensis and Mauremys mutica , which has been implicitly or explicitly assumed by a number of previous authors on the basis of morphological data. Instead, M. annamensis was indicated to be closest to Mauremys i versoni , a species assumed to be most divergent among the East Asian Mauremys by previous authors.     

Seasonal changes in abundance and shifts in dominance of life history stages of the carrageenophyte Sarcothalia crispata (Rhodophyta, Gigartinales) in south central Chile

The population dynamics of the carrageenophyte Sarcothalia crispatais described from subtidal beds at two localities in south-central Chile. Seasonal fluctuations in total density and biomass were not evident. Frondswere identified to phase by the presence of reproductive structures and theresorcinol reaction. The monthly changes in abundance of each kind offrond were determined. Permanent gametophytic or sporophyticdominance was not evident: the more exposed site showed a seasonal shiftfrom sporophytic dominance in summer to gametophytic dominance inwinter, whereas the more protected site showed an interannual shift fromgametophytic to sporophytic dominance. The differences between localitiesand years suggest a very local population dynamics with large contributionof self-seeding to the maintenance of the S. crispata beds.     

Enzyme specificity under dynamic control II: Principal component analysis of α-lytic protease using global and local solvent boundary conditions

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of alpha-lytic protease. The wild-type alpha-lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190-->Ala binding pocket mutant has a much broader specificity, actively hydrolyzing substrates ranging from Ala to Phe. Based on a combination of multiconformation analysis of cryo-X-ray crystallographic data, solution nuclear magnetic resonance (NMR), and normal mode calculations, we had hypothesized that the large alteration in specificity of the mutant enzyme is mainly attributable to changes in the dynamic movement of the two walls of the specificity pocket. To test this hypothesis, we performed a principal component analysis using 1-nanosecond molecular dynamics simulations using either a global or local solvent boundary condition. The results of this analysis strongly support our hypothesis and verify the results previously obtained by in vacuo normal mode analysis. We found that the walls of the wild-type substrate binding pocket move in tandem with one another, causing the pocket size to remain fixed so that only small substrates are recognized. In contrast, the M190A mutant shows uncoupled movement of the binding pocket walls, allowing the pocket to sample both smaller and larger sizes, which appears to be the cause of the observed broad specificity. The results suggest that the protein dynamics of alpha-lytic protease may play a significant role in defining the patterns of substrate specificity. As shown here, concerted local movements within proteins can be efficiently analyzed through a combination of principal component analysis and molecular dynamics trajectories using a local solvent boundary condition to reduce computational time and matrix size.