Protein folding simulation with solvent-induced force field: folding pathway ensemble of three-helix-bundle proteins

We propose a coarse-grained model of proteins that take into account solvent effects and apply it for simulating folding of a three-helix-bundle protein. The energy functional form, refined from our previous work (Takada et al., J Chem Phys 1999;110:11616-11629), tries to closely imitate real physico-chemical interactions. In particular, the hydrogen bond that depends on local dielectric constant, the helix capping effect, and side-chain entropic effects are included. With use of the model, we simulate folding of the GA module of an albumin binding domain, 1prb(7-53), finding most trajectories reach at the native topology within 1 micros. In the simulation, helices 1 and 3 are mostly formed earlier accompanied by non-specific collapse, while second helix is intrinsically less stable and is formed with the help of tertiary contacts at later stage. We compute an analog of the transition state ensemble and compare it with those of other three-helix-bundle proteins. The transition state of 1prb(7-53) includes a few specific tertiary contacts of C terminus of helix 3 with the loop region between helices 1 and 2. This resembles, but is not equivalent to, an early formed region of fragment B of staphylococcal protein A, but is quite different from the folding transient structures of a de novo designed three-helix-bundle peptide.     

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.     

Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5

Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.     

Expression pattern of the Brachyury gene in the arrow worm paraspadella gotoi (chaetognatha)

Arrow worms (the phylum Chaetognatha), which are among the major marine planktonic animals, are direct developers and exhibit features characteristic of both deuterostomes and protostomes. In particular, the embryonic development of arrow worms appears to be of the deuterostome type. Brachyury functions critically in the formation of the notochord in chordates, whereas the gene is expressed in both the blastopore and stomodeum invagination regions in embryos of hemichordates and echinoderms. Here we analyzed the expression of Brachyury (Pg-Bra) in the arrow worm Paraspadella gotoi and showed that Pg-Bra is expressed in the blastopore region and the stomodeum region in the embryo and then around the mouth opening region at the time of hatching. The expression of Pg-Bra in the embryo resembles that of Brachyury in embryos of hemichordates and echinoderms, whereas that in the mouth opening region in the hatchling appears to be novel.     

Model of the alphaLbeta2 integrin I-domain/ICAM-1 DI interface suggests that subtle changes in loop orientation determine ligand specificity

The interaction of the alphaLbeta2 integrin with its cellular ligand the intercellular adhesion molecule-1 (ICAM-1) is critical for the tight binding interaction between most leukocytes and the vascular endothelium before transendothelial migration to the sites of inflammation. In this article we have modeled the alphaL subunit I-domain in its active form, which was computationally docked with the D1 domain of the ICAM-1 to probe potential protein-protein interactions. The experimentally observed key interaction between the carboxylate of Glu 34 in the ICAM-1 D1 domain and the metal ion-dependent adhesion site (MIDAS) in the open alphaL I-domain was consistently reproduced by our calculations. The calculations reveal the nature of the alphaLbeta2/ICAM-1 interaction and suggest an explanation for the increased ligand-binding affinity in the "open" versus the "closed" conformation of the alphaL I-domain. A mechanism for substrate selectivity among alphaL, alphaM, and alpha2 I-domains is suggested whereby the orientation of the loops within the I-domain is critical in mediating the interaction of the Glu 34 carboxylate of ICAM-1 D1 with the MIDAS.     

Natural<Superscript>15</Superscript> N Abundance of Plants and Soil N in a Temperate Coniferous Forest

Measurement of nitrogen isotopic composition (¹⁵N) of plants and soil nitrogen might allow the characteristics of N transformation in an ecosystem to be detected. We tested the measurement of ¹⁵N for its ability to provide a picture of N dynamics at the ecosystem level by doing a simple comparison of ¹⁵N between soil N pools and plants, and by using an existing model. ¹⁵N of plants and soil N was measured together with foliar nitrate reductase activity (NRA) and the foliar NO₃^– pool at two sites with different nitrification rates in a temperature forest in Japan. ¹⁵N of plants was similar to that of soil NO₃^– in the high-nitrification site. Because of high foliar NRA and the large foliar NO₃^– pool at this site, we concluded that plant ¹⁵N indicated a great reliance of plants on soil NO₃^– there. However, many ¹⁵N of soil N overlapped each other at the other site, and ¹⁵N could not provide definitive evidence of the N source. The existing model was verified by measured ¹⁵N of soil inorganic N and it explained the variations of plant ¹⁵N between the two sites in the context of relative importance of nitrification, but more information about isotopic fractionations during plant N uptake is required for quantitative discussions about the plant N source. The model applied here can provide a basis to compare ¹⁵N signatures from different ecosystems and to understand N dynamics.     

Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class Ib molecules

The vomeronasal organ (VNO) of the mouse has two neuronal compartments expressing distinct families of pheromone receptors, the V1Rs and the V2Rs. We report here that two families of major histocompatibility complex (MHC) class Ib molecules, the M10 and the M1 families, show restricted expression in V2R-expressing neurons. Our data suggest that neurons expressing a given V2R specifically co-express one or a few members of the M10 family. Biochemical and immunocytochemical analysis demonstrates that in VNO sensory dendrites M10s belong to large multi-molecular complexes that include pheromone receptors and beta2-microglobulin (beta2m). In cultured cells, M10s appear to function as escort molecules in transport of V2Rs to the cell surface. Accordingly, beta2m-deficient mice exhibit mislocalization of V2Rs in the VNO and a specific defect in male-male aggressive behavior. The functional characterization of M10 highlights an unexpected role for MHC molecules in pheromone detection by mammalian VNO neurons.