Protein secondary structure and stability determined by combining exoproteolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability.     

Volume and heat capacity of model non-aqueous self-assembly systems

Blends of immiscible polymers are often stabilized by block copolymers which can form non-aqueous micelles and microemulsions in the liquid polymers. The phase diagrans, apparent volumes and apparent heat capacities of model non-aqueous binary and ternary systems were studied in order to investigate the conditions under which such self-assembly systems could form. 1,2-Hexanediol, which can cosolubilize hexane and ethyleneglycol, forms inverse micelles in hexane and weak microaggregates in ethyleneglycol. Genapol X-060, a commercial alcoholic surfactant containing on the average an aliphatic chain of 13 carbons and 6 oxyethylenes (C₁₃E₆), forms microaggregates in poly(ethyleneglycol) 400. These self-assembly systems are strengthen in the presence of a third component which has an affinity for the inner phase.Presented at the Symposium, 76^th CSC Congress, Sherbrooke, Quebec, May 30–June 3, 1993, honoring Professor Donald Patterson on the occasion of his 65^th birthday.     

Performance of SuSi: a method for generating atomistic models of amorphous polymers based on a random search of energy minima

The performance of a recently developed method to generate representative atomistic models of amorphous polymers has been investigated. This method, which is denoted SuSi, can be defined as a random generator of energy minima. The effects produced by different parameters used to define the size of the system and the characteristics of the generation algorithm have been examined. Calculations have been performed on poly(L,D-lactic) acid (rho = 1.25 g/cm3) and nylon 6 (rho = 1.084 g/cm(3)), which are important commercial polymers.     

Polymer-Titanium hybrids obtained by radical polymerization and Sol-Gel process

The possibility to prepare hybrids made by poly(vinyl acetate) (PVAc), poly(methyl methacrylate) (PMMA) and/or poly(ethyl acrylate) (PEtA) with TiO₂ was studied. The processes of polymer formation-radical polymerization and sol-gel process for inorganic network —were achieved simultaneously. Due to a high reactivity of titanium isopropoxide (TIP) in the sol-gel process, a complexant comonomer, allyl acetoacetate (AlAcAc), was used. Covalent bonds between polymer and inorganic chains were obtained by addition of trialkoxysilane derivates with vinyl (VTES) or methacryloyl (MPTS) groups. The presence of TIP inhibits the radical polymerization of vinyl acetate (VAc). The PVAc-TiO₂ hybrids were produced by the sol-gel process of TIP in the presence of pre-obtained PVAc. Except for VTES and MPTS, trialkoxysilane derivates with methyl (MeTES), octyl (OTES) and phenyl (PTES) groups were used. The thermal stability of hybrids is strongly affected by TiO₂ presence and by the type of trialkoxysilane derivates. The thermal stability of PVAc hybrids decreases in the presence of TiO₂ inorganic network. The glass transition temperature of polymers increases in the presence of the inorganic network.     

The influence of cosolvent polarity on the flow properties of hydroalcoholic gels: empirical models

The aim of this study was to investigate the influence of cosolvent polarity on the rheological flow properties of aqueous and hydroalcoholic gels obtained from the Carbopol Ultrez 10 base and used for topical applications. Specifically, we have examined the effect of pH (range 4.0--7.0) on the consistency and flow properties of dispersed systems in water and mixtures--15 : 85% v/v of methanol : water, ethanol : water, n-propanol : water and n-butanol : water--at a constant polymer concentration of 0.3% w/w. The gels, which had decreasing polarity values in the jellifying medium, showed qualitatively similar flow behavior, characteristic of pseudoplastic systems, and all of the flow curves were adjusted to the Ostwald model. Sigmoidal dose response functions were calculated to describe the flow and consistency indexes as a function of pH. As a result, the influence of alcohol polarity on the polymer network has been assessed meaningfully using the empirical parameters obtained: maximum consistency index value (k(max)), pH value required for 50% development of polymer network (pH(50)), and asymptotic flow index value (n(min)) for the fully structured gels.     

Droplet evaporation study applied to DNA chip manufacturing

DNA chips are potentially powerful technologies for genotyping and gene expression profiling that rely on comparative analyses of up to thousands of "spots of analysis" on a glass support. The spot quality throughout the support influences spot-to-spot variations within an array and the repeatability of data across experiments. For glass slide DNA microarrays, droplets of DNA solution are deposited on functionalized glass slides and left to react through complete evaporation of the droplet. On hydrophobic flat surfaces, different modes of droplet evaporation can be attained. Under atmospheric pressure, water droplets tend to evaporate under two main regimes. Initially, the droplet flattens with a constant contact area, and then the droplet shrinks at a constant contact angle. As a result, the diameter and morphology of thousands of spots on microarrays are not uniform. This leads to poor and unreliable data processing results. In this work, we report the evaporation of an aqueous solution under a constant contact area mode. Evaporation under reduced pressure and the effect of reagent additives to the solution have been investigated. Video microscopy and digital image analysis techniques were applied to monitor the evaporation of the droplets. A mixture of surfactants was developed to maintain a constant area regime during evaporation and to form homogeneous spots. The control of some physicochemical properties (wetting, evaporation rate) of the droplet allows the formation of well-controlled spots compatible with DNA grafting. The influence of surfactant molecules on the mechanisms of evaporation is also discussed.     

Influence of the coordination number of nitrogen on the structure of 6aλ4-thia-1,3,4,6-tetraazapentalenic systems. Crystal structure analyses of 3-(2-pyridylimino)-3H-[1,2,4]thiadiazolo[4,3-a]pyridine and its 1-methylated salt

Structural data were obtained by X-ray crystallography for the title compounds which show that they are essentially planar and exhibit an approximately linear N2-S1-N8 arrangement. In compound 3 the separation between the sulfur atom and the pyridine nitrogen atom (2.61 Å) is larger than the Huggins constant energy distance (2.58 Å), suggesting that there is little or no bonding between them. The methylated salt 4 , on the contrary, has a closer S…N(pyridine) distance (2.19 Å) with an estimated bond dissociation energy of 6 kcal/mole.     

Crystal structure oftrans-ethyl(1,5,6-trimethylbenzimidazole)-bis(dimethylglyoximato)cobalt(III). Relationships between structural and spectroscopic properties in compounds of the general formulae [Co(dmgH)2(R)(1,5,6-Me3Bzm)]

Summary The synthesis and x-ray crystal structure oftrans-[Co(dmgH)₂(Et)(1,5,6-Me₃Bzm)] where dmgH=dimethylglyoximate(–1), and 1,5,6-Me₃Bzm=1,5,6-trimethylbenzimidazole, is reported. The compound C₁₉H₂₆N₆O₄Co is monoclinic, space group P2₁/n;a=11.700(4);b=24.205(6);c=8.500(3) Å and =101.63(3)°. D(calcd) 1.299 g cm^–3; Z=4 and R=0.066 for 2359 independent reflections. Comparison of Co-N(axial ligand) bond lengths for compounds of general formulaetrans-[Co(dmgH)₂(R)(L)], with L=pyridine or 1,5,6-trimethylbenzimidazole and R=CH(CN)Cl, CH₂NO₂, Me, Et,i-Pr, cyclo-hexyl or adamantyl is made. The Co–N(1,5,6-Me₃Bzm) bond lengths of the trimethylbenzimidazole derivatives show a fairly linear relationship with the electronic parameter of the axial R group, derived from the¹³C-n.m.r. spectra of their pyridine analogues. The influence of steric effects on the properties of these Co^III compounds is discussed.     

Oscillating autoxidation reaction of p-xylene in the liquid phase, catalyzed by metal oxides

Oscillatory reaction was observed during liquid-phase oxidation of p-xylene in the presence of certain transition metal oxides as catalysts. Experimental conditions under which oscillations occur and parameters influencing this behavior are described.

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Acidity and activity of H-mordenite in m-xylene isomerization

The dependence of the activity of H-mordenites prepared from a natural mordenite on the nature and strength of the acid groups present was studied. The superior catalytic activity and stability of the H(Ag)–MOR zeolite compared with other H-forms is explained on the basis of NH₃ adsorption and IR measurements.

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