Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes, A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality, For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties, The imidazole function was conjugated to these CS-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid), The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments, 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates, In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates, The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.

Lightly branched comb polyesters: application in fast drying solvent-borne coating formulations

Novel oxazoline-based comb-polymers possessing linoleyl or oleic side chains have been synthesized and used to produce low viscosity coatings. Inclusion of the polymers in model paint formulations results in coatings that exhibit faster drying times than commercially available alkyd resin formulations. The comb polymers were produced from diol substituted oxazoline monomers that were synthesized through a scalable, solvent free protocol and purified by simple recrystallisation. Co-polymerisation of the oxazolines with adipic acid at 160 °C in the bulk resulted in the targeted polyester comb type polymers. The polymers were soluble in a range of organic solvents and compatible with commercial alkyd resins. Model paint formulations containing up to 40 wt% of the linoleyl-based comb polymers exhibited a dramatic reduction in viscosity (from 35 to 13 Poise at 25 °C) with increasing quantities of polymer added. Dynamic mechanical analysis (DMA) studies revealed that the drying rate of the model paint formulations containing the comb polymers was enhanced when compared with that of commercial alkyd resins.

Determination of orientations of aromatic groups in self-assembled peptide fibrils by polarised Raman spectroscopy

In this paper we describe a novel combination of Raman spectroscopy, isotope editing and X-ray scattering as a powerful approach to give detailed structural information on aromatic side chains in peptide fibrils. The orientation of the tyrosine residues in fibrils of the peptide YTIAALLSPYS with respect to the fibril axis has been determined from a combination of polarised Raman spectroscopy and X-ray diffraction measurements. The Raman intensity of selected tyrosine bands collected at different polarisation geometries is related to the values and orientation of the Raman tensor for those specific vibrations. Using published Raman tensor values we solved the relevant expressions for both of the two tyrosine residues present in this peptide. Ring deuteration in one of the two tyrosine side chains allowed for the calculation to be performed individually for both, by virtue of the isotopic shift that eliminates band overlapping. Sample disorder was taken into account by obtaining the distribution of orientations of the samples from X-ray diffraction experiments. The results provide previously unavailable details about the molecular conformation of this peptide, and demonstrate the value of this approach for the study of amyloid fibrils.

Influence of elastase on alanine-rich peptide hydrogels

The self-assembly of the alanine-rich amphiphilic peptides Lys(Ala)6Lys (KA6K) and Lys(Ala)6Glu (KA6E)with homotelechelic or heterotelechelic charged termini respectively has been investigated in aqueous solution. These peptides contain hexa-alanine sequences designed to serve as substrates for the enzyme elastase. Electrostatic repulsion of the lysine termini in KA6K prevents self-assembly, whereas in contrast KA6E is observed, through electron microscopy, to form tape-like fibrils, which based on X-ray scattering contain layers of thickness equal to the molecular length. The alanine residues enable efficient packing of the side-chains in a beta-sheet structure, as revealed by circular dichroism, FTIR and X-ray diffraction experiments. In buffer, KA6E is able to form hydrogels at sufficiently high concentration. These were used as substrates for elastase, and enzyme-induced de-gelation was observed due to the disruption of the beta-sheet fibrillar network. We propose that hydrogels of the simple designed amphiphilic peptide KA6E may serve as model substrates for elastase and this could ultimately lead to applications in biomedicine and regenerative medicine.

Enzymatically-triggered, isothermally responsive polymers: re-programming poly(oligoethylene glycols) to respond to phosphatase

Polymers which can respond to externally applied stimuli have found much application in the biomedical field due to their (reversible) coil–globule transitions. Polymers displaying a lower critical solution temperature are the most commonly used, but for blood-borne (i.e., soluble) biomedical applications the application of heat is not always possible, nor practical. Here we report the design and synthesis of poly(oligoethylene glycol methacrylate)-based polymers whose cloud points are easily varied by alkaline phosphatase-mediated dephosphorylation. By fine-tuning the density of phosphate groups on the backbone, it was possible to induce an isothermal transition: A change in solubility triggered by removal of a small number of phosphate esters from the side chains activating the LCST-type response. As there was no temperature change involved, this serves as a model of a cell-instructed polymer response. Finally, it was found that both polymers were non cytotoxic against MCF-7 cells (at 1 mg·mL–1), which confirms promise for biomedical applications.

(1)H, (15)N and (13)C assignments of the Rhipicephalus (Boophilus) microplus anti-microbial peptide microplusin

Microplusin, a Rhipicephalus (Boophilus) microplus anti-microbial peptide (AMP) is the first member of a new family of cysteine-rich AMPs with histidine-rich regions at the N- and C-termini, which is being fully characterized by biophysical and biochemical methods. Here we report the NMR resonance assignments for (1)H, (15)N, and (13)C nuclei in the backbone and side chains of the microplusin as basis for further studies of structure, backbone dynamics and interactions mapping.

Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations

Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 angstrom resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis, for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting beta-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.

The Synthesis and Characterization of Bicyclooxacalixarenes

Bicyclooxacalixarenes were synthesized in high yield via a selective, room temperature Sn Ar reaction of phluoroglucinol with 2,6-dichloropyridines. Functionality on the 2,6-dichloropyridioe was varied by changing the electron-withdrawing groups in the 3 and 5 positions (using chlorine, nitro groups, and cyano groups) and the side-chains in the 4-position (using ethyl, butyl, phenyl and ρ-tolyl groups). The resulting cage-like molecules were studied by X-ray crystallography and tested for metal complexation.

New metallomolecular wires based on carbon-rich bridging systems: a systematic study

This dissertation presents and discusses the preparation of molecular wires (MW) candidates that would then be probed for electron transfer properties. These wires are bridged by 1,4-diethynylbenzene derivatives with alkoxy side chains with palladium and ruthenium metal complex termini. Characterization of these compounds was performed by usual spectroscopic techniques like 1H, 13C{1H} and 31P{1H} NMR, MS, FTIR and UV-Vis as well as by cyclic voltammetry which allowed classifying the candidates in the Robin–Day system and determination of bridges side chain and length effects on electronic transport. Preparation of the 1,4-diethynylbenzene derivatives was done with synthetic pathways that relied heavily in palladium catalyzed cross-couplings (Sonogashira). A family of single ringed 1,4-diethynylbenzene ligands with different length alkoxy side chains (OCH3, OC2H5, OC7H15) was thus prepared allowing for the influence of these ring decorations to be assessed. The ruthenium binuclear rods showed communication between metal centres only when the shorter ligands were used whereas the longer Ru complexes showed only one redox pair in CV studies which is in agreement to non-communicating metal centres. Cyclic voltammetry studies show irreversible one wave processes for palladium dinuclear complexes, making these rods function as molecular insulators. Fluorescence decay studies performed on the prepared compounds (ligands and complexes) show a pattern of decreasing decay times upon coordination to the metal centres which can due to ligand charge redistribution upon coordination leading to non-radiative relaxation paths. Regarding the X-ray structures, two new ligand related structures were obtained as well as new structure for a palladium rod. The effect of the side chains was observed to be important to the wires’ electronic properties when comparing with the analogues without a side chain. The effect brought by longer chains is nevertheless almost negligible.

Purification and structural characterisation of (1 -> 3 ; 1 -> 6)-beta-D-glucans (botryosphaerans) from Botryosphaeria rhodina grown on sucrose and fructose as carbon sources: a comparative study

Two botryosphaerans, exopolysaccharides (EPS) secreted by the ascomyceteous fungus Botryosphaeria rhodina, when grown on sucrose and fructose as sole carbon sources, were structurally compared after their isolation from the culture medium. Both EPS were submitted to trypsin digestion, and eluted as a single peak on gel filtration. Total acid hydrolysis yielded only glucose, and data from methylation analysis and Smith degradation indicated that both EPS constituted a main chain of glucopyranosyl beta(1 -> 3) linkages substituted at O-6. The products obtained after partial acid hydrolysis demonstrated side chains consisting of glucosyl- and gentiobiosyl- linked beta(1 -> 6) residues. C-13-NMR spectroscopy studies showed that all glucosidic linkages were of the beta-configuration. The carbon source affected the side chain structures of botryosphaeran but not the main chain makeup. Sucrose produced less branching (21%) than fructose (31%). (c) 2005 Published by Elsevier Ltd.

The shielding effect of glycerol against protein ionization in electrospray mass spectrometry

Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the signal of horse heart myoglobin. The signal suppression was probably caused by the interaction of glycerol molecules with the proteins to create a shielding effect that prevents the ionization of the basic and/or acidic groups in the amino acid side chains. To overcome this difficulty the glycerol concentration was decreased to 5% (v/v) by dialyzing the Taq polymerase solution against water, and the cone voltage in the ESI triple-quadrupole mass spectrometer was set at 80-130 V. This permitted observation of a mass spectrum that contained ions corresponding to protonation of up to 50% of the ionizable basic groups. In the absence of glycerol up to 85% of the basic groups of Taq polymerase became ionized, as observed in the mass spectrum at relatively low cone voltages. An explanation of these and other observations is proposed, based on strong interactions between the protein molecules and glycerol. For purposes of comparison similar experiments were performed on myoglobin, a small protein with 21 basic groups, whose ionization was apparently suppressed in the presence of 1% (v/v) glycerol, since no mass spectrum could be obtained even at high cone voltages. Copyright (C) 2003 John Wiley Sons, Ltd.

Analyzing glycerol-mediated protein oligomerization by electrospray ionization mass spectrometry

Glycerol is widely used as protein stabilizer, in both local and commercial preparations, so it has become necessary to develop methods for mass spectrometric analysis of protein preparations in the presence of glycerol. However, this stabilizing agent may cause signal suppression when present in high concentrations, and is also known to induce protein supercharging even at low concentrations. This work reports the,use of electrospray ionization (ESI) mass spectrometry to characterize glycerol-mediated protein oligomerization. this phenomenon seems to involve the formation of strong non-covalent interactions between protein and glycerol involving close contact between the monomers, leading to formation of protein oligomers adducted with glycerol molecules under the characteristic analytical conditions of the ESI interface. At high orders of oligomerization a lower number of glycerol molecules is required to maintain the high oligomeric states than for the dimers and trimers, and it is possible that for the higher oligomers the monomers become so close to one another that non-covalent bonds between the side chains of the amino acid residues in the proteins may be established. Copyright (C) 2005 John Wiley & Sons, Ltd.

Profiling the peptidome of the venom from the social wasp Agelaia pallipes pallipes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of genetic algorithms and solvation potential to study peptides structure

In this work, genetic algorithms concepts along with a rotamer library for proteins side chains and implicit solvation potential are used to optimize the tertiary structure of peptides. We starting from the known PDB structure of its backbone which is kept fixed while the side chains allowed adopting the conformations present in the rotamer library. It was used rotamer library independent of backbone and a implicit solvation potential. The structure of Mastoporan-X was predicted using several force fields with a growing complexity; we started it with a field where the only present interaction was Lennard-Jones. We added the Coulombian term and we considered the solvation effects through a term proportional to the solvent accessible area. This paper present good and interesting results obtained using the potential with solvation term and rotamer library. Hence, the algorithm (called YODA) presented here can be a good tool to the prediction problem. (c) 2007 Elsevier B.V. All rights reserved.

Using genetic algorithm to design protein sequence

In this work, genetic algorithms concepts along with a rotamer library for proteins side chains are used to optimize the tertiary structure of the hydrophobic core of Cytochrome b(562) starting from the known PDB structure of its backbone which is kept fixed while the side chains of the hydrophobic core are allowed to adopt the conformations present in the rotamer library. The atoms of the side chains forming the core interact via van der Waals energy. Besides the prediction of the native core structure, it is also suggested a set of different amino acid sequences for this core. Comparison between these new cores and the native are made in terms of their volumes, van der Waals energies values and the numbers of contacts made by the side chains forming the cores. This paper proves that genetic algorithms area efficient to design new sequence for the protein core. (C) 2007 Elsevier B.V. All rights reserved.