Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). II. Semi-empirical energy calculations.

The molecular conformation of the basic pancreatic trypsin inhibitor (BPTI) is known in considerable detail from both X-ray studies in single crystals and NMR studies in solution. The NMR experiments showed that the aromatic rings of the phenylalanyl and tyrosyl residues can undergo rapid rotational motions about the C beta--Cv bond. The present paper describes a model investigation of the mechanistic aspects of these intramolecular rotational motions. From calculations of the conformational energies for molecular species derived from the X-ray structure by rotations of individual aromatic rings, it was apparent that the rotational motions of the aromatics could only be understood in a flexible structure. Flexibility was simulated by allowing the protein to relax to an energetically favorable conformation for each of the different rotation states of the aromatic rings. It was then of particular interest to investigate how the perturbations caused by different rotation states of the aromatic rings were propagated in the protein structure. It was found that the rotation axes C beta--Cv were only slightly affected (delta X1 approximately less than 20 degrees. The most sizeable perturbations are caused by through space interactions with nearby atoms, which move away from the ring center and thus release the steric hindrance opposing the rotational motions. The values for the energy barriers obtained from the energy minimization are of the same order of magnitude as those measured by NMR.     

The primary structure of the hemoglobin of the electric eel (Electrophorus electricus)

The blood of the Electric Eel contains only one hemoglobin component. The primary structures of the alpha- and beta-chains are presented. These were separated by high-performance liquid chromatography, using a new kind of buffer system. The alpha-chains are acetylated, and consist of 142 residues, while the beta-chains are not blocked, and consist of 147 residues. The phylogenetic distances between these and the alpha- and beta-chains of human hemoglobin are 48 and 50% amino-acid exchanges, respectively. The relationship between primary structure and the Bohr effect and Root effect is discussed, especially the significance of the serine found in position F9 beta.     

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Structural and Functional Analysis of the Natural JNK1 Inhibitor Quercetagetin

c-Jun NH2-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3-K) play critical roles in chronic diseases such as cancer, type II diabetes, and obesity. We describe here the binding of quercetagetin (3,3′,4′,5,6,7-hydroxyflavone), related flavonoids, and SP600125 to JNK1 and PI3-K by ATP-competitive and immobilized metal ion affinity-based fluorescence polarization assays and measure the effect of quercetagetin on JNK1 and PI3-K activities. Quercetagetin attenuated the phosphorylation of c-Jun and AKT, suppressed AP-1 and NF-κB promoter activities, and also reduced cell transformation. It attenuated tumor incidence and reduced tumor volumes in a two-stage skin carcinogenesis mouse model.Our crystallographic structure determination data show that quercetagetin binds to the ATP-binding site of JNK1. Notably, the interaction between Lys55, Asp169, and Glu73 of JNK1 and the catechol moiety of quercetagetin reorients the N-terminal lobe of JNK1, thereby improving compatibility of the ligand with its binding site. The results of a theoretical docking study suggest a binding mode of PI3-K with the hydroxyl groups of the catechol moiety forming hydrogen bonds with the side chains of Asp964 and Asp841 in the p110γ catalytic subunit. These interactions could contribute to the high inhibitory activity of quercetagetin against PI3-K. Our study suggests the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases.     

2G12-Expressing B Cell Lines May Aid in HIV Carbohydrate Vaccine Design Strategies

The highly conserved cluster of high-mannose glycans on the HIV-1 envelope glycoprotein, gp120, has been highlighted as a target for neutralizing antibodies. 2G12, the first HIV-1 antiglycan neutralizing antibody described, binds with an unusual domain-exchanged structure that creates a high-affinity multivalent binding surface. It is an interesting challenge for rational vaccine design to generate immunogens capable of eliciting domain-exchanged 2G12-like responses. We recently showed that di-mannose recognition by the variable domains of 2G12 is independent of domain exchange but that exchange is critical for virus neutralization. Carbohydrate-based immunogens aimed at inducing 2G12-like antibodies may need to drive both di-mannose recognition and domain exchange through interactions with B cell receptors. Here we assessed the ability of such immunogens to activate mouse B cell lines displaying domain-exchanged wild-type 2G12 (2G12 WT), a non-domain-exchanged Y-shaped variant (2G12 I19R), and germ line 2G12 (2G12 gl). We show that several immunogens, including heat-killed yeast and bacteria, can activate both 2G12 WT and 2G12 I19R B cells. However, only discrete clusters of high-mannose glycans, as on recombinant forms of the HIV-1 envelope trimer and oligodendrons, activate 2G12 WT B cells. Furthermore, no immunogen tested activated 2G12 gl cells. Our results support the hypothesis that in order to drive domain exchange of an antimannose antibody response, a boost with an immunogen displaying discrete clusters of high-mannose glycans not recognized by conventional Y-shaped antibodies will be required. Additionally, a molecule capable of activating 2G12 gl cells might also be required. The results highlight broadly neutralizing antibody-expressing mouse B cells as potentially useful tools for carbohydrate immunogen screening.     

Combustion influences on natural abundance nitrogen isotope ratio in soil and plants following a wildfire in a sub-alpine ecosystem

This before-and-after-impact study uses the natural abundance N isotope ratio (δ¹⁵N) to investigate the effects of a wildfire on sub-alpine ecosystem properties and processes. We measured the ¹⁵N signatures of soil, charred organic material, ash and foliage in three sub-alpine plant communities (grassland, heathland and woodland) in south-eastern Australia. Surface bulk soil was temporarily enriched in ¹⁵N immediately after wildfire compared to charred organic material and ash in all plant communities. We associated the enrichment of bulk soil with fractionation of N during combustion and volatilization of N, a process that also explains the sequential enrichment of ¹⁵N of unburnt leaves > ash > charred organic material in relation to duration and intensity of heating. The rapid decline in ¹⁵N of bulk soil to pre-fire values indicates that depleted ash, containing considerable amounts of total N, was readily incorporated into the soil. Foliar δ¹⁵N also increased with values peaking 1 year post-fire. Foliar enrichment was foremost coupled with the release of enriched NH₄ ⁺ into the soil owing to isotopic discrimination during volatilization of soluble N and combustion of organic material. The mode of post-fire regeneration influenced foliar ¹⁵N enrichment in two species indicating use of different sources of N following fire. The use of natural abundance of ¹⁵N in soil, ash and foliage as a means of tracing transformation of N during wildfire has established the importance of combustion products as an important, albeit temporary source of inorganic N for plants regenerating after wildfire.     

Three-Dimensional Protein Fold Determination from Backbone Amide Pseudocontact Shifts Generated by Lanthanide Tags at Multiple Sites

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