Polymorphic Self-Organization of Lauroyl Peptide in Response to pH and Concentration.

Amphipathic peptides are attractive building blocks for the preparation of self-assembling, bio-inspired, and stimuli responsive nanomaterials with pharmaceutical interest. The bioavailability of these materials can be improved with the insertion of d amino acid residues to avoid fast proteolysis in vivo. With this knowledge, a new lauroyl peptide consisting of a sequence of glycine, glycine, d-serine, and d-lysine was designed. In spite of its simple sequence, this lipopeptide self-assembles into spherical micelles at acid pH, when the peptide moiety adopts disordered conformations. Self-aggregates reshape toward fibers at basic pH, following the conformational transition of the peptide region from random coil to ß-sheet. Finally, hydrogels are achieved at basic pH and higher concentrations. The transition from random coil to ß-sheet conformation of the peptide headgroup obtained by increasing pH was monitored by circular dichroism and vibrational spectroscopy. A structural analysis, performed by combining dynamic light scattering, small-angle X-ray scattering, transmission electron microscopy, and molecular dynamic simulations, demonstrated that the transition allows the self-assemblies to remodel from spherical micelles to rodlike shapes, to long fibers with rectangular cross-section and a head-tail-tail-head structure. The viscoelastic behavior of the hydrogels formed at the highest pH was investigated by rheology measurements.

Switchable length nanotubes from a self-assembling pH and thermosensitive linear l,d-peptide-polymer conjugate.

Preparation and characterization of a pH and thermosensitive linear l,d-octapeptide-poly(dimethylamino ethyl methacrylate) ((l-Val-d-Val)4-PDMAEMA) conjugate is reported. The hydrophobic uncharged linear (l-Val-d-Val)4 octapeptide was designed to self-assemble in nanotubes by exploiting the tubular self-assembling properties of linear peptides with regularly alternating enantiomeric sequences. pH and thermosensitive PDMAEMA was obtained by atom transfer radical polymerization (ATRP). The conjugate was prepared by click-chemistry on the solid phase synthetized peptide. Because of the strong interactions between the peptide moieties, long single channel nanotubes (0.2-1.5 µm) are formed also at acidic pH with the fully charged polymer. At 25 °C and basic pH the size of the nanotubes did not change significantly. In basic conditions and temperature above the PDMAEMA lower critical solution temperature (LCST) a significant increase of the length of the nanotubes up to several micrometers is observed. The size is retained for several days after cooling back to room temperature. Sonication significantly reduces the nanotube length (0.2-0.5 µm) forming low polydisperse nanotubes. The elongation of the nanotubes is fully reversible by restoring acidic pH. This is the first example, to our knowledge, of thermosensitive peptide-polymer single channel nanotubes with length that can be varied from hundreds of nanometers to several micrometers.

Oxidized Buckypaper for Stir-Disc Solid Phase Extraction: Evaluation of Several Classes of Environmental Pollutants Recovered from Surface Water Samples.

This paper describes, for the first time, the use of oxidized buckypaper (BP) as a sorbent membrane of a stir-disc solid phase extraction module. The original device, consisting of a BP disc ( d = 34 mm) enveloped in a polypropylene mesh pouch, was designed to extract organic micropollutants (OMPs) from environmental water samples in dynamic mode. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to analyze the extracts. Several classes of pesticides and pharmaceuticals were chosen as model compounds to evaluate key parameters affecting the recovery rates. To this end, the effects of adsorption time, desorption time, stirring speed, type and volume of solvent, and sample volume were thoroughly examined. After optimization, a novel and in-depth study was conducted to find a correlation between physicochemical properties of the analytes and extraction yields. Recoveries were mainly governed by a combination of log P and p Ka values. As indicated, hydrophilic compounds with log P < 1 showed poor affinity for the oxidized BP, compounds having log P > 1 exhibited recoveries ranging between 50% and 100% depending on their p Ka, while compounds with p Ka between 6 and 7.5 gave low yields irrespective of their log P. The analytical method was also validated and tested as large scale screening method of OMPs in surface waters. The analysis of real samples revealed the presence of some nonsteroidal anti-inflammatory drugs, sulfonamides, and pesticides at low ng L-1 concentration levels with relative standard deviations lower than 8%.

Liquid chromatography/mass spectrometry identification of intermediates and vulcanization products by using squalene as vulcanization model compound.

RATIONALE: Sulfur-vulcanized rubber is a three-dimensional polymer network, insoluble in all organic solvents. For this reason, vulcanization products are difficult to study and identify by conventional analytical techniques. To simplify this task, low molecular weight olefins have been used as model compounds (MCs) in place of rubber in vulcanization experiments. METHODS: In this work, the vulcanization process was investigated using squalene (SQ) as MC. By-products, intermediates and products were separated by semipreparative reversed-phase liquid chromatography (RPLC) with UV detection. Each fraction was collected, concentrated and characterized by flow injection analysis (FIA) and non-aqueous reversed-phase (NARP) LC coupled to positive atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Under the latter conditions, an Information-Dependent Acquisition (IDA) was performed on a linear ion trap mass spectrometer to obtain structural information. RESULTS: Several vulcanized compounds containing up to three SQ molecules, cross-linked with chains involving up to 14 sulfur atoms overall, have been identified along with some of their oxidized products (epoxides and hydroperoxides). The FIA-MS spectra showed peak clusters, each of which included two-three subclusters; the interpretation was complicated by the occurrence of more ion species per product, by the unsaturation grade and by the characteristic isotopic distribution of sulfur. The enhanced product ion scan (EPI) spectra, acquired during the IDA experiments, supported the FIA-MS identification allowing one to count the number of sulfur atoms. CONCLUSIONS: The sensitivity of the developed analytical strategy was due to the enrichment factor achieved via semipreparative chromatography and the very good response of the APCI detection. Pattern fragmentation and chromatographic behavior simplified the identification of the cured compounds and their oxidized products, whose occurrence was related to the grade of oxidation of SQ used as reagent. Copyright © 2016 John Wiley & Sons, Ltd.

Geometrical, conformational and topological restraints in regular nucleosome compaction in chromatin.

The folding of the nucleosome array into a chromatin fiber modulates DNA accessibility and is therefore an important factor for the control of gene expression. The statistical analysis of the nucleosome repeat length in chromatin fibers reveals the presence of a ten-fold periodicity suggesting the existence of orientational constraints of the nucleosome units that provide the geometrical conditions of helical conformations. Recently, the elucidation of the x-ray crystal structure of a nucleosome tetramer array and the interpretation of electron microscopy images of reconstituted nucleosome arrays suggested two different architectures of the chromatin fiber. We approached the problem by integrating the experimental findings with geometrical, conformational and topological restraints, under the hypothesis of the minimum distortion of the nucleosome and linker DNA structures. We show that the excluded volume at linker crossing and the torsional energy limit the possible close packing of the nucleosomes in the chromatin fiber. In particular, the torsional energy of the chromatin fiber appears crucial in determining the kind of nucleosome packing for short nucleosome repeat lengths as in telomeres and yeast chromatin.

Prediction of nucleosome positioning in genomes: limits and perspectives of physical and bioinformatic approaches.

Nucleosomes, the fundamental repeating subunits of all eukaryotic chromatin, are responsible for packaging DNA into chromosomes inside the cell nucleus and controlling gene expression. While it has been well established that nucleosomes exhibit higher affinity for select DNA sequences, until recently it was unclear whether such preferences exerted a significant, genome-wide effect on nucleosome positioning in vivo. For this reason, an increasing interest is arising on a wide-ranging series of experimental and computational analyses capable of predicting the nucleosome positioning along genomes. Toward this goal, we propose a theoretical model for predicting nucleosome thermodynamic stability in terms of DNA sequence. Based on a statistical mechanical approach, the model allows the calculation of the sequence-dependent canonical ensemble free energy involved in nucleosome formation. The theoretical free energies were evaluated for 90 single nucleosome DNA tracts and successfully compared with those obtained with nucleosome competitive reconstitution. These results, obtained for single nucleosomes, could in principle allow the calculation of the intrinsic affinity of nucleosomes along DNA sequences virtually opening the possibility of predicting the nucleosome positioning along genomes on physical basis. The theoretical nucleosome distribution was compared and validated with that of yeast and human genome experimentally determined. The results interpret on a physical basis the experimental nucleosome positioning and are comparable with those obtained adopting models based on the identification of some recurrent sequence features obtained from the statistical analysis of a very large pool of nucleosomal DNA sequences provided by the positioning maps of genomes.

A statistical thermodynamic approach for predicting the sequence-dependent nucleosome positioning along genomes.

Nucleosomes are the fundamental repeating unit of chromatin and constitute the structural building blocks of the eukaryotic genome. The distribution of nucleosomes along the genome is a significant aspect of chromatin structure and influences gene regulation through modulation of DNA accessibility. For this reason, an increasing interest is arising in models capable of predicting the nucleosome positioning along genomes. Toward this goal, we propose a theoretical model for predicting nucleosome thermodynamic stability in terms of DNA sequence. The model, based on a statistical mechanical approach allows the calculation of the canonical ensemble free energy involved in nucleosome formation. The theoretical free energies were evaluated for about one hundred nucleosome DNA tracts and successfully compared with those obtained in different laboratories with nucleosome competitive reconstitution (correlation coefficient equal to 0.92). We extended these results to the nucleosome positioning along genomes. To test our model, the theoretical nucleosome distribution was compared with that of yeast genome experimentally determined. The results are comparable with those obtained by different authors adopting models based on identifying some recurrent sequence features obtained from the statistical analysis of a very large pool of nucleosomal DNA sequences provided by the positioning maps of genomes.

Peptides with regular enantiomeric sequences: a wide class of modular self-assembling architectures.

Organic trans-annular assemblies constitute an expanding class of structures with promising applications for the design of nanotechnological devices. Among the strategies developed for the engineering of organic nanotubes, those characterized by regular alternating enantiomeric amino acid sequences have been proven particularly useful. In fact, cyclic peptides with an even number of regularly alternating D- and L-amino acids have the tendency to adopt local beta-conformation that are capable of forming trans-annular self-assembling architectures, hydrogen bond directed. The formation of such structures is the result of the conformational equivalence of the monomer units, a general principle that associates stereo-chemical to chemical equivalence in a polymer chain. For configurationally alternating sequences the conformational equivalence produces cyclic structures, where a monomer unit is related to the adjacent along the chain by a roto-reflection axis, Sn. A slight relaxation of the conformational equivalence can formally transform a cyclic structure into a conformationally quasi-equivalent helical structures characterized by the presence of polar inner channels, which allow the transient binding for an activated flow of specific ions. To prove our early predictions, we synthesized different alternating polypeptide and the corresponding linear and cyclic oligopeptides and investigated their conformations by NMR and CD spectroscopy as well as the formation of self-assembling structures by increasing the concentration in solution. Moreover, their predicted ability to behave as an ion-channel across bilayer membranes are investigated and experimental evidence of single molecule conducting events are reported. Finally, the possibility is suggested to obtain self-assembled trans-annular structures by chemically bridging the amino acid side chains stabilized using different strategies. A complex construct with good perspective for nano-technological applications is proposed in which cyclic DL-lysine side chains are bridged by the formation of salycilaldimmine metal chelates.

A statistical approach for analyzing structural and regulative information in prokaryotic genomes.

Although DNA is iconized as a straight double helix, it does not exist in this canonical form in biological systems. Instead, it is characterized by sequence dependent structural and dynamic deviations from the monotonous regularity of the canonical B-DNA. Despite the complexity of the system, we showed that DNA structural and dynamics large-scale properties can be predicted starting from the simple knowledge of nucleotide sequence by adopting a statistical approach. The paper reports the statistical analysis of large pools of different prokaryotic genes in terms of the sequence-dependent curvature and flexibility. Conserved features characterize the regions close to the Start Translation Site, which are related to their function in the regulation system. In addition, regular patterns with three-fold periodicity were found in the coding regions. They were reproduced in terms of the nucleotide frequency expected on the basis of the genetic code and the pertinent occurrence of the aminoacid residues.

A simple procedure to weight empirical potentials in a fitness function so as to optimize its performance in ab initio protein-folding problem.

In an approach to the protein folding problem by a Genetic Algorithm, the fitness function plays a critical role. Empirical potentials are generally used to build the fitness function, and they must be weighted to obtain a valuable one. The weights are generally found by the comparison with a set of misfolded structures (decoys), but a dependence of the obtained fitness generally arises on the used decoys. Here we describe a general procedure to find out, from a given set of potentials, their better linear combination that could either identify the wild structure or prove their powerlessness. We use topological considerations over the hyperspace of the potentials, and a multiple linear inequalities solver. The iterated method flows through the following steps: it determines a direction in the hyperspace of the potentials, which identifies the native structure as a vertex among a set of misfolded decoys. A multiple linear inequalities solver obtains the direction. A Genetic Algorithm, tailored to the specific problem, uses the fitness function defined by this direction and generally reaches a new structure better than the experimental one, which is added to the ensemble. The decoys so generated are not dependent on a deterministic criterion. This iterative procedure can be stopped either by identifying an effective fitness function or by proving the impossibility of its achievement. In order to test the method under the hardest conditions, we choose numerous and heterogeneous quantities as components of the fitness function. This method could be a useful tool for the scientific community in order to test any fitness proposed and to recognize the most important components on which it is built.