Temperature dependence of the free energy landscape of the src-SH3 protein domain

The temperature dependence of the free energy landscape of the src-SH3 protein domain is investigated through fully atomic simulations in explicit solvent. Simulations are performed above and below the folding transition temperature, enabling an analysis of both protein folding and unfolding. The transition state for folding and unfolding, identified from the free energy surfaces, is found to be very similar, with structure in the central hydrophobic sheet and little structure throughout the rest of the protein. This is a result of a polarized folding (unfolding) mechanism involving early formation (late loss) of the central hydrophobic sheet at the transition state. Unfolding simulations map qualitatively well onto low-temperature free energy surfaces but appear, however, to miss important features observed in folding simulations. In particular, details of the folding mechanism involving the opening and closing of the hydrophobic core are not captured by unfolding simulations performed under strongly denaturing conditions. In addition, free energy surfaces at high temperatures do not display a desolvation barrier found at lower temperatures, involving the expulsion of water molecules from the hydrophobic core.     

Encapsulated <Emphasis Type="Italic">Escherichia coli</Emphasis> in alginate beads capable of secreting a heterologous pectin lyase

Background  

Production of heterologous proteins in the E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process.     

Spatial genome organization

The linear sequence of genomes exists within the three-dimensional space of the cell nucleus. The spatial arrangement of genes and chromosomes within the interphase nucleus is nonrandom and gives rise to specific patterns. While recent work has begun to describe some of the positioning patterns of chromosomes and gene loci, the structural constraints that are responsible for nonrandom positioning and the relevance of spatial genome organization for genome expression are unclear. Here we discuss potential functional consequences of spatial genome organization and we speculate on the possible molecular mechanisms of how genomes are organized within the space of the mammalian cell nucleus.     

Substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae

We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-β,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.     

Reconstructing a chloride-binding site in a bacterial neurotransmitter transporter homologue

In ion-coupled transport proteins, occupation of selective ion-binding sites is required to trigger conformational changes that lead to substrate translocation. Neurotransmitter transporters, targets of abused and therapeutic drugs, require Na(+) and Cl(-) for function. We recently proposed a chloride-binding site in these proteins not present in Cl(-)-independent prokaryotic homologues. Here we describe conversion of the Cl(-)-independent prokaryotic tryptophan transporter TnaT to a fully functional Cl(-)-dependent form by a single point mutation, D268S. Mutations in TnaT-D268S, in wild type TnaT and in serotonin transporter provide direct evidence for the involvement of each of the proposed residues in Cl(-) coordination. In both SERT and TnaT-D268S, Cl(-) and Na(+) mutually increased each other's potency, consistent with electrostatic interaction through adjacent binding sites. These studies establish the site where Cl(-) binds to trigger conformational change during neurotransmitter transport.     

To be, or not to be two sites: that is the question about LeuT substrate binding

Transport proteins of the neurotransmitter sodium symporter (NSS) family regulate the extracellular concentration of several neurotransmitters in the central nervous system. The only member of this family for which atomic-resolution structural data are available is the prokaryotic homologue LeuT. This protein has been used as a model system to study the molecular mechanism of transport of the NSS family. In this Journal Club, we discuss two strikingly different LeuT transport mechanisms: one involving a single high-affinity substrate binding site and one recently proposed alternative involving two high-affinity substrate binding sites that are allosterically coupled.     

Human embryonal epithelial cells of the developing small intestinal crypts can express the Hodgkin-cell associated antigen Ki-1 (CD30) in spontaneous abortions during the first trimester of gestation

Background  

Ki-1 (CD30) antigen expression is not found on peripheral blood cells but its expression can be induced in vitro on T and B lymphocytes by viruses and lectins. Expression of CD30 in normal tissues is very limited, being restricted mainly to a subpopulation of large lymphoid cells; in particular, cells of the recently described anaplastic large cell lymphoma (ALCL), the Reed-Sternberg (RS) cells of Hodgkin's lymphoma and scattered large parafollicular cells in normal lymphoid tissues. More recent reports have described CD30 expression in non-hematopoietic and malignant cells such as cultured human macrophages, human decidual cells, histiocytic neoplastic cells, mesothelioma cells, embryonal carcinoma and seminoma cells.     

Early post-fire regeneration of a Pinus halepensis forest on Mount Párnis,Greece

Abstract. The post-fire regeneration of a 45-yr-old Pinus halepensis (Aleppo pine) forest, burned in July 1989, has been studied on Mount Párnis, Attiki, Greece. Four experimental plots at various slopes and exposures were established at altitudes of 400 - 450 m, and monitored for 3 yr at 3-month intervals. Early regeneration took place abundantly, through both resprouting and seed germination of mostly hard-seeded herbs and shrubs; the floristic richness was high with 80 taxa. Pine seedling emergence took place during the winter of the first post-fire year. The mean pine seedling density by the end of the recruitment period (March 1990) was 5–6 seedlings/m². This density decreased slightly during late spring and considerably during summer. During the second post-fire year only a relatively slight decline was observed; thereafter the density was stabilized to 1 - 2 seedlings/m². Mortality follows a negative exponential curve that levels off at ca. 20 %. Height distributions throughout the three post-fire years were all positively skewed as a result of the presence of few very tall saplings. A considerable fraction (20 %) of very short (5–15 cm) saplings were still alive 39 months after the fire; these may constitute the sapling bank. Based on the analysis of height distribution curves, it is concluded that the taller seedlings survived significantly better than the shorter ones.