Amphipols from A to Z.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

Validation of an automatic comet assay analysis system integrating the curve fitting of combined comet intensity profiles.

In recent years, the single-cell gel electrophoresis (comet) assay has become a reference technique for the assessment of DNA fragmentation both in vitro and in vivo at the cellular level. In order to improve the throughput of genotoxicity screening, development of fully automated systems is clearly a must. This would allow us to increase processing time and to avoid subjectivity brought about by frequent manual settings required for the 'classical' analysis systems. To validate a fully automatic system developed in our laboratory, different experiments were conducted in vitro on murine P388D1 cells with increasing doses of ethyl methanesulfonate (up to 5 mM), thus covering a large range of DNA damage (up to 80% of DNA in the tail). The present study (1) validates our 'in house' fully automatic system versus a widely used semi-automatic commercial system for the image-analysis step, and versus the human eye for the image acquisition step, (2) shows that computing tail DNA a posteriori on the basis of a curve fitting concept that combines intensity profiles [G. Dehon, P. Bogaerts, P. Duez, L. Catoire, J. Dubois, Curve fitting of combined comet intensity profiles: a new global concept to quantify DNA damage by the comet assay, Chemom. Intell. Lab. Syst. 73 (2004) 235-243] gives results not significantly different from the 'classical' approach but is much more accurate and easy to undertake and (3) demonstrates that, with these increased performances, the number of comets to be scored can be reduced to a minimum of 20 comets per slide without sacrificing statistical reliability.

Investigation of the action patterns of pectinmethylesterase isoforms through kinetic analyses and NMR spectroscopy. Implications In cell wall expansion.

Well characterized pectin samples were incubated with cell wall-bound and -solubilized pure isoforms of pectinmethylesterase from mung bean hypocotyls (Vigna radiata). Both enzyme activity and average product structure were determined at intervals along the deesterification pathway at pH 5.6 and 7.6. The latter analyses were performed by 13C NMR spectroscopy, and the degree of esterification was probed by both 13C NMR and potentiometric measurements. A dichotomy was observed in the behavior of the alpha and gamma isoforms when compared with that of the beta isoenzyme. Ideal blockwise deesterification mechanisms reproduced the experimental average structures (methylester distribution) throughout the course of the reaction. In the case of the alpha and gamma isoforms, a single chain mechanism associated with a free carboxyl group at the second nearest neighbor position could be postulated at pH 5.6, whereas some multiple attack character was required to reproduce the data at pH 7.6. Several mechanisms that differed from the preceding ones were compatible with the data for the beta isoform at the two pH values. Both the nature of the polysaccharides produced in these reactions and the role of pectinmethylesterase in the cell wall-stiffening process along the growth gradient are discussed.

An efficient procedure for studying pectin structure which combines limited depolymerization and 13C NMR.

A protocol for partial thermally-induced depolymerization of differently methoxylated pectin samples is described. The resulting macromolecules have been fully characterized with various complementary techniques, such as size exclusion chromatography (SEC), potentiometry, viscometry and 13C NMR. Optimum conditions afford samples at 50-80% yield with weight-average molecular weights in the 4 to 20 kDa range. The major fraction of these polysaccharides adopts the random-coil conformation and such samples are suitable for 13C NMR structural studies at room temperature. The methoxyl distributions of two apple pectin samples with a degree of esterification (DE) between 54 and 74% and a citrus pectin (DE, 72%) were shown to be random in nature, whereas that of a lightly methoxylated apple pectin (DE 39%) was partially blockwise. The carbon relaxation parameters of the depolymerized pectins attain asymptotic values for Mw > 4 kDa. The Mw values estimated from intrinsic viscosity data with the Mark-Houwink relationship reported for native pectins are in good agreement with those obtained by either end-group analysis (NMR) or SEC. Thus, all the physicochemical data indicate that the secondary structure of the isolated chains of depolymerized pectin is closely related to that of the parent polymers. Finally, pectinmethylesterase activity towards the depolymerized pectins was similar to that of the untreated samples.

NMR analysis of carbohydrates with model-free spectral densities: the dispersion range revisited.

Over the past decade molecular mechanics and molecular dynamics studies have demonstrated considerable flexibility for carbohydrates. In order to interpret the corresponding NMR parameters, which correspond to a time-averaged or 'virtual' conformer, it is necessary to simulate the experimental data using the averaged geometrical representation obtained with molecular modelling methods. This structural information can be transformed into theoretical NMR data using empirical Karplus-type equations for the scalar coupling constants and the appropriate formalism for the relaxation parameters. In the case of relaxation data, the 'model-free' spectral densities have been widely used in order to account for the internal motions in sugars. Several studies have been conducted with truncated model-free spectral densities based on the assumption that internal motion is very fast with respect to overall tumbling. In this report we present experimental and theoretical evidence that suggests that this approach is not justified. Indeed, recent results show that even in the case of moderate-sized carbohydrates internal motions are occurring on the same timescale as molecular reorientation. Simulations of relaxation parameters (NOESY volumes, proton cross-relaxation rates, carbon T1 and nOe values) in the dispersion range (0.1 < tau(c) < 5 ns) show that rates of internal motion can be fairly precisely defined with respect to overall tumbling. Experimental data for a variety of oligosaccharides clearly indicate similar timescales for internal and overall motion.