Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding

The gamma-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.     

Enhanced rates of P700<Superscript>+</Superscript> dark-reduction in leaves of<Emphasis Type="Italic"> Cucumis sativus</Emphasis> L. photoinhibited at chilling temperature

The changes in electron transport within photosystem I (PSI) were studied in detached leaves of Cucumis sativus L. during the course of irradiation with moderate white light (300 mol photons m^–2 s^–1) at 4°C. When intact leaves were exposed to the combination of moderate light and low temperature, the amplitude of far-red light-induced P700 absorbance changes at 820 nm (A₈₂₀), a relative measure of PSI, progressively decreased as the light treatment time increased. Almost no oxidation of P700 was noticeable after 5 h. Methyl viologen accelerated the oxidation of P700 to a steady-state level and also increased the magnitudes of A₈₂₀ changes in photoinhibited leaves, reflecting the rapid removal of electrons from native carriers. Photoinhibition under moderate light and chilling temperature also accelerated the rate of P700⁺ reduction after far-red light excitation as the half-times of the two exponential components of P700⁺ decay curves decreased relative to the control ones. A detailed analysis of the kinetics of P700⁺ reduction using diuron alone or the combination of diuron and methyl viologen strongly favours an increased rate of electron donation from stromal reductants to PSI through the plastoquinone pool following photoinhibitory treatment. Importantly, the marked acceleration of P700⁺ re-reduction is the consequence of the irradiation of leaf segments at low temperature and not caused by chilling stress alone.Abbreviations A ₀ and A ₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - FR Far-red light - F _X , F _A , and F _B Iron–sulfur centers - MT Multiple-turnover flash - MV Methyl viologen - Ndh NAD(P)H-dehydrogenase - PQ Plastoquinone - PS Photosystem - P700 Reaction-center chlorophyll of PSI - ST Single-turnover flash     

Automated analysis of vapor diffusion crystallization drops with an X-ray beam

Crystallogenesis, usually based on the vapor diffusion method, is currently considered one of the most difficult steps in macromolecular X-ray crystallography. Due to the increasing number of crystallization assays performed by protein crystallographers, several automated analysis methods are under development. Most of these methods are based on microscope images and shape recognition. We propose an alternative method of identifying protein crystals: by directly exposing the crystallization drops to an X-ray beam. The resulting diffraction provides far more information than classical microscope images. Not only is the presence of diffracting crystals revealed, but also a first estimation of the space group, cell parameters, and mosaicity is obtained. In certain cases, it is also possible to collect enough data to verify the presence of a specific substrate or a heavy atom. All these steps are performed without the sometimes tedious necessity of removing crystals from their crystallization drop.     

Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation processes

A comprehensive view of the physiological state of Escherichia coli cells at the completion of fermentation processes for biopharmaceutical production was attained via two-dimensional gel electrophoretic analysis of cellular proteins. For high cell density fermentations in which phosphate is depleted to induce recombinant protein expression from the alkaline phosphatase promoter, proteome analysis confirms that phosphate limitation occurs. Known phosphate starvation inducible proteins are observed at high levels; these include the periplasmic phosphate binding protein and the periplasmic phosphonate binding protein. The phn (EcoK) locus of these E. coli K-12 strains remains cryptic, as demonstrated by failure to grow with phosphonate as the sole phosphorus source. Proteome analysis also provided evidence that cells utilize alternative carbon and energy sources during these fermentation processes. To address regulatory issues in the biopharmaceutical industry, comparative electrophoretic analyses were conducted on a qualitative basis for four different fermentation processes. Using this approach, the protein profiles for these processes were found to be highly similar, with the vast majority (85-90%) of proteins detected in all profiles. The observed similarity in proteomes suggests that multiproduct host cell protein immunoassays are a feasible means of quantifying host-derived polypeptides from a variety of biopharmaceutical fermentation processes.     

Pitx genes in Tunicates provide new molecular insight into the evolutionary origin of pituitary

We have initiated a project aimed at documenting molecular and cellular changes underlying the emergence of the hypothalamo-hypophyseal axis in Chordates. Considering the phylogenetic position of Tunicates and the 'pan-hypophyseal' expression pattern of Pitx genes in Vertebrate pituitary, we searched for a Pitx-related homeobox gene in the ascidian Ciona intestinalis, and identified Ci-Pitx (ona intestinalis uitary homeobo gene). We also isolated Cs-Pitx and Bs-Pitx, the Ci-Pitx respective counterparts of Ciona savignyi and Botryllus schlosseri, two other Tunicate species. Ci-Pitx mRNA encodes a putative protein exhibiting the diagnostic K50-Paired-class homeodomain and a conserved C-terminal Aristaless domain. Embryonic expression pattern of Ci-Pitx revealed a conserved expression domain in the anterior neural ridge and subsequently in the pharyngeal primordium, defined in Vertebrates as the stomodeal ectomere, which encompasses the presumptive pituitary territory. This shows that expression at early steps of pituitary development is a feature of Pitx-related genes that was already present in the last common ancestor of Chordates.