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.     

Influence of temperature and humidity on insecticidal effect of three diatomaceous earth formulations against larger grain borer (Coleoptera: Bostrychidae)

Laboratory experiments were carried out to evaluated three diatomaceous earth (DE) formulations--Protect-It, PyriSec (at dose rates 500, 1000, and 1500 ppm), and DEA-P (at dose rates 75, 150, and 500 ppm)--against the larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrychidae), adults in stored maize, Zea mays L., at three temperatures (20, 25, and 30 degrees C) and two relative humidity (RH) levels (55 and 75%). At these conditions, the capability of progeny production in the treated substrate also was assessed. Adult survival was high, at all doses of Protect-It and PyriSec. Progeny production was also high. In contrast with the other two DEs, DEA-P was highly effective and caused complete mortality to the exposed P. truncatus adults, even at the lowest dose rate (75 ppm). In addition, progeny production was completely suppressed. Generally, Protect-it and PyriSec were more effective at 20 degrees C than at 30 degrees C. In contrast, the efficacy of DEA-P was continuously high in all temperatures and relative humidities examined.     

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.     

Improvement of tablet coating uniformity using a quality by design approach

A combination of analytical and statistical methods is used to improve a tablet coating process guided by quality by design (QbD) principles. A solid dosage form product was found to intermittently exhibit bad taste. A suspected cause was the variability in coating thickness which could lead to the subject tasting the active ingredient in some tablets. A number of samples were analyzed using a laser-induced breakdown spectroscopy (LIBS)-based analytical method, and it was found that the main variability component was the tablet-to-tablet variability within a lot. Hence, it was inferred that the coating process (performed in a perforated rotating pan) required optimization. A set of designed experiments along with response surface modeling and kriging method were used to arrive at an optimal set of operating conditions. Effects of the amount of coating imparted, spray rate, pan rotation speed, and spray temperature were characterized. The results were quantified in terms of the relative standard deviation of tablet-averaged LIBS score and a coating variability index which was the ratio of the standard deviation of the tablet-averaged LIBS score and the weight gain of the tablets. The data-driven models developed based on the designed experiments predicted that the minimum value of this index would be obtained for a 6% weight gain for a pan operating at the highest speed at the maximum fill level while using the lowest spraying rate and temperature from the chosen parametric space. This systematic application of the QbD-based method resulted in an enhanced process understanding and reducing the coating variability by more than half.     

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.     

Effect of temperature and commodity on insecticidal efficacy of spinosad dust against Sitophilus oryzae (Coleoptera: Curculionidae) and Rhyzopertha dominica (Coleoptera: Bostrychidae)

The insecticidal effect of spinosad dust, a formulation that contains 0.125% spinosad, was evaluated against adults of Sitophilus oryzae (L.) and Rhyzopertha dominica (F.) at three temperature levels (20, 25, and 30 degrees C) and four commodities (wheat, Triticum aestivum L.; barley, Hordeum vulgare L.; rice, Oryza sativa L.; and maize, Zea mays L.). For this purpose, quantities of the above-mentioned grains were treated with spinosad at two dose rates (20 and 50 ppm of the formulation, corresponding to 0.025 and 0.06 ppm AI, respectively), and mortality of the exposed adults in the treated grains was measured after 7 and 14 d, whereas progeny production was assessed 65 d later. Generally, for both species, mortality increased with dose, exposure interval, and temperature. For S. oryzae, adult survival and progeny production were lower on wheat than the other grains. After 14 d of exposure, mortality of S. oryzae adults on wheat treated with 50 ppm ranged between 61 and 98%, whereas in the other three commodities it did not exceed 42%. Mortality of R. dominica after 14 d on grains treated 50 ppm ranged between 91 and 100%. For this species, progeny production from exposed parental adults was low in all commodities regardless of temperature. Results indicate that spinosad dust can be used as an alternative to traditional grain protectants, but its effectiveness is highly determined by the target species, commodity, dose, and temperature.     

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.