Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [¹⁵N]NH₄Cl and [¹³C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly ¹³C,¹⁵N-labeled protein secreted by approximately 10¹⁰D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional ¹H-¹³C HSQC spectrum confirms ¹³C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.     

Multi-domain, cell-envelope proteinases of lactic acid bacteria

The multi-domain, cell-envelope proteinases encoded by the genes prtB of Lactobacillus delbrueckii subsp. bulgaricus, prtH of Lactobacillus helveticus, prtP of Lactococcus lactis, scpA of Streptococcus pyogenes and csp of Streptococcus agalactiae have been compared using multiple sequence alignment, secondary structure prediction and database homology searching methods. This comparative analysis has led to the prediction of a number of different domains in these cell-envelope proteinases, and their homology, characteristics and putative function are described. These domains include, starting from the N-terminus, a pre-pro-domain for secretion and activation, a serine protease domain (with a smaller inserted domain), two large middle domains A and B of unknown but possibly regulatory function, a helical spacer domain, a hydrophilic cell-wall spacer or attachment domain, and a cell-wall anchor domain. Not all domains are present in each cell-envelope proteinase, suggesting that these multi-domain proteins are the result of gene shuffling and domain swapping during evolution.     

A surface acoustic wave sensor functionalized with a polypyrrole molecularly imprinted polymer for selective dopamine detection

A surface acoustic wave sensor operating at 104 MHz and functionalized with a polypyrrole molecularly imprinted polymer has been designed for selective detection of dopamine (DA). Optimization of pyrrole/DA ratio, polymerization and immersion times permitted to obtain a highly selective sensor, which has a sensitivity of 0.55°/mM (≈550 Hz/mM) and a detection limit of ≈ 10 nM. Morphology and related roughness parameters of molecularly imprinted polymer surfaces, before and after extraction of DA, as well as that of the non imprinted polymer were characterized by atomic force microscopy. The developed chemosensor selectively recognized dopamine over the structurally similar compound 4‐hydroxyphenethylamine (referred as tyramine), or ascorbic acid,which co‐exists with DA in body fluids at a much higher concentration. Selectivity tests were also carried out with dihydroxybenzene, for which an unexpected phase variation of order of 75% of the DA one was observed. Quantum chemical calculations, based on the density functional theory, were carried out to determine the nature of interactions between each analyte and the PPy matrix and the DA imprinted PPy polypyrrole sensing layer in order to account for the important phase variation observed during dihydroxybenzene injection. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparative analysis of the methods used for finding surface energy to investigate protein interaction behavior on chromatographic supports

Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.     

Rapid degradation of pyrogenic carbon

Pyrogenic carbon (PC‐ charcoal, biochar or black carbon) represents a poorly understood component of the global carbon (C) cycle, but one that has considerable potential to mitigate climate change through provision of long‐term soil C sequestration. Mass balance calculations suggest global PC production and stocks are not in balance, indicating a major gap in our understanding of the processes by which PC is re‐mineralized. We collected PC samples derived from the same wood material and exposed to natural environmental conditions for 1 and 11 years. We subjected these materials to repeated laboratory incubation studies at temperatures of up to 60 °C, as ground surface temperatures above 30 °C and up to 60 °C occur regularly over a significant area of the tropics and sub‐tropics. Mineralization rates were not different for the two samples and followed an exponential Arrhenius function that suggest an average turnover time of 67 years for conditions typical of a tropical savannah environment. Microbial biomass as measured by chloroform fumigation and DNA extractions was the same for the two samples, but abiotic CO₂ production was lower for the fresh PC sample than that for the aged sample. Nuclear magnetic resonance spectroscopy, hydrogen pyrolysis and scanning electron microscopy demonstrate that the measured CO₂ production originates dominantly from polycyclic aromatic compounds rather than any minor labile components. Therefore, rapid, sub‐centennial rates of re‐mineralization of PC on the soil surface in tropical and sub‐tropical environments may represent a major and hitherto unidentified mechanism for balancing the PC production at the global scale.     

Cortical bone growth and dietary stress among subadults from Nubia's Batn el Hajar

Cortical bone growth is analyzed for 174 children from a Medieval Christian population at Kulubnarti in the Batn el Hajar of Sudanese Nubia (550–1450 AD ). Using the tibia as a representative long bone, total subperiosteal area, cortical area, medullary area, and percent cortical area at midshaft were calculated. While growth in total and cortical areas, as well as in length, appear to be fairly well maintained, percent cortical area reveals unusual growth patterns which reflect excessive endosteal resorption. Compared to the relative reduction in bone mass which has been observed in malnourished living children, as well as with previously reported evidence for stress in the Kulubnarti population, the present data support an interpretation of nutritionally related stress and of no major diachronic dietary change.     

All-Atom Structural Models for Complexes of Insulin-Like Growth Factors IGF1 and IGF2 with Their Cognate Receptor

Type 1 insulin-like growth factor receptor (IGF1R) is a membrane-spanning glycoprotein of the insulin receptor family that has been implicated in a variety of cancers. The key questions related to molecular mechanisms governing ligand recognition by IGF1R remain unanswered, partly due to the lack of testable structural models of apo or ligand-bound receptor complexes. Using a homology model of the IGF1R ectodomain IGF1RΔβ, we present the first experimentally consistent all-atom structural models of IGF1/IGF1RΔβ and IGF2/IGF1RΔβ complexes. Our explicit-solvent molecular dynamics (MD) simulation of apo-IGF1RΔβ shows that it displays asymmetric flexibility mechanisms that result in one of two binding pockets accessible to growth factors IGF1 and IGF2, as demonstrated via an MD-assisted Monte Carlo docking procedure. Our MD-generated ensemble of structures of apo and IGF1-bound IGF1RΔβ agrees reasonably well with published small-angle X-ray scattering data. We observe simultaneous contacts of each growth factor with sites 1 and 2 of IGF1R, suggesting cross-linking of receptor subunits. Our models provide direct evidence in favor of suggested electrostatic complementarity between the C-domain (IGF1) and the cysteine-rich domain (IGF1R). Our IGF1/IGF1RΔβ model provides structural bases for the observation that a single IGF1 molecule binds to IGF1RΔβ at low concentrations in small-angle X-ray scattering studies. We also suggest new possible structural bases for differences in the affinities of insulin, IGF1, and IGF2 for their noncognate receptors.     

Fab is the most efficient format to express functional antibodies by yeast surface display

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.