Dielectric and dynamic mechanical study of the mobility of poly(t-butylacrylate) chains in diblock copolymers: polystyrene-b-poly(t-butylacrylate)

A calorimetric, dielectric and dynamic-mechanical study of the dynamics of the poly(t-butyl acrylate) (PtBa) chains has been carried out in a PtBa homopolymer and two polystyrene (PS)-b-PtBa block copolymers with different PtBa chain lengths. The DSC results show that the size of the cooperative rearranging regions is similar in the homopolymers and the copolymers, both for the PtBa rich- and the PS-rich regions. Therefore, no significant contributions are found arising from composition fluctuations in the copolymers. The relaxation map obtained from dielectric relaxation indicates that there are no differences in the temperature dependence of the α-relaxation of the PtBa block in the three samples studied. However, there are larger differences for the values obtained from DMTA experiments. Contrary to the α-relaxation, the relaxation map for the β-transition shows that the characteristic times for the PtBa blocks are smaller in the homopolymer than in the copolymers. In principle, these are unexpected results because the β-relaxations have a more local character than the α-ones. The width of the α-relaxation increases with T for all the samples, and it is slightly larger for the copolymers. The intensity of the α-relaxation is larger (between 3 and 4 times) for the homopolymer. Considering the molecular weights of the PtBa blocks, this effect has to be ascribed to the existence of frozen amorphous PtBa due to the existence of the glassy PS domains in the microphase separated copolymers. Molecular Dynamic Simulations (MDSs) for different sequences of the polymers under study were carried out. The conformational analysis was carried out between 1000 and 1700 K. The analysis of the variation of angles 1 and 2 of the ester group of PtBa points out the existence of a correlation between the conformational changes of the side group of the polymer chains and their relaxational behaviour

Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake

Indexación: Web of Science.Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.http://nanoscalereslett.springeropen.com/articles/10.1186/s11671-016-1260-

Gating of a pH-Sensitive K2P Potassium Channel by an Electrostatic Effect of Basic Sensor Residues on the Selectivity Filter

K+ channels share common selectivity characteristics but exhibit a wide diversity in how they are gated open. Leak K2P K+ channels TASK-2, TALK-1 and TALK-2 are gated open by extracellular alkalinization. The mechanism for this alkalinization-dependent gating has been proposed to be the neutralization of the side chain of a single arginine (lysine in TALK-2) residue near the pore of TASK-2, which occurs with the unusual pKa of 8.0. We now corroborate this hypothesis by transplanting the TASK-2 extracellular pH (pHo) sensor in the background of a pHo-insensitive TASK-3 channel, which leads to the restitution of pHo-gating. Using a concatenated channel approach, we also demonstrate that for TASK-2 to open, pHo sensors must be neutralized in each of the two subunits forming these dimeric channels with no apparent cross-talk between the sensors. These results are consistent with adaptive biasing force analysis of K+ permeation using a model selectivity filter in wild-type and mutated channels. The underlying free-energy profiles confirm that either a doubly or a singly charged pHo sensor is sufficient to abolish ion flow. Atomic detail of the associated mechanism reveals that, rather than a collapse of the pore, as proposed for other K2P channels gated at the selectivity filter, an increased height of the energetic barriers for ion translocation accounts for channel blockade at acid pHo. Our data, therefore, strongly suggest that a cycle of protonation/deprotonation of pHo-sensing arginine 224 side chain gates the TASK-2 channel by electrostatically tuning the conformational stability of its selectivity filter

Mutation Arg336 to Lys in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase originates an enzyme with increased oxaloacetate decarboxylase activity

AbstractSaccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes one of the first reactions in the biosynthesis of carbohydrates. Apart from the physiologically important reaction, the enzyme also presents low oxaloacetate decarboxylase and pyruvate kinase-like activities. Data from the crystalline structure of homologous Escherichia coli PEP carboxykinase suggest that Arg333 may be involved in stabilization of enolpyruvate, a postulated reaction intermediate. In this work, the equivalent Arg336 from the S. cerevisiae enzyme was changed to Lys or Gln. Kinetic analyses of the varied enzymes showed that a positive charge at position 336 is critical for catalysis of the main reaction, and further suggested different rate limiting steps for the main reaction and the secondary activities. The Arg336Lys altered enzyme showed increased oxaloacetate decarboxylase activity and developed the ability to catalyze pyruvate enolization. These last results support the proposal that enolpyruvate is an intermediate in the PEP carboxykinase reaction and suggest that in the Arg336Lys PEP carboxykinase a proton donor group has appeared

The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region

Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (β/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-β-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (β/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (β/α)8-barrel domain