La directive

Master [120] en droit, Université catholique de Louvain, 201

Tetrapeptidbasiertes Proteindesign - ein Lösungsansatz für das inverse Proteinfaltungsproblem

Das Ziel dieser Arbeit war die Entwicklung eines neuartigen Algorithmus zum Proteindesign basierend auf kurzen Peptidfragmenten. Die Methode verwendet statistische Informationen über Tetrapeptidkonformationen. Hierfür wurde eine modifizierte Datenaufbereitung entwickelt, die in der Lage war, die nichtredundante Ausgangsdatenbasis für solche statistische Analysen zu vergrößern. Es wurden verschiedene, nichthomologe Aminosäuresequenzen errechnet, welche in die artifizielle Struktur des Proteins TOP7 (Kuhlman et al., Science, 2003; 302:1364-1368) falten sollten. Zwei der designten Proteine, M5 und M7, wurden rekombinant hergestellt und mittels Fluoreszensspektroskopie, Circulardicroismus und NMR charakterisiert. Die Ergebnisse zeigen Hinweise auf geordnete Tertiärstrukturen und kooperative Faltungs/Entfaltungsübergänge. Die beiden neuen Proteine weisen weiterhin eine sehr hohe Stabilität gegen Temperatur- und Denaturanz-induzierte Entfaltung auf.The aim of this work was the development of a new algorithm for protein design based on short peptide fragments. The method uses statistical information on tetrapeptide backbone conformations. We created a modified data selection procedure which was able to extend the nonredundant data basis available for statistical analysis. A series of non homologous polypeptide sequences were created that were predicted to fold into the artificial structure of the protein TOP 7 (Kuhlman et al., Science, 2003; 302:1364-1368). Two of the designed proteins, M5 and M7, were expressed and characterized by fluorescence spectroscopy, circular dichroism and NMR. They showed the hallmarks of well-ordered tertiary structure as well as cooperative folding/unfolding transitions. The two novel proteins were found to be highly stable against temperature and denaturant-induced unfolding.von Roman Dallüg

Fast comprehensive two-dimensional gas chromatography (GC x GC) using 50-mu m ID second-dimension columns

Up till now, comprehensive two-dimensional gas chromatography has suffered from the serious disadvantage that the first-dimension separation has to be operated both at sub-optimum flow and at slow temperature programming rates to enable the required number of modulations across a first-dimension peak. Consequently, the separation efficiency of the first-dimension is (far) below optimum and the overall analysis times become longer than necessary. This problem can be solved by selecting a short 50-μm ID second-dimension column, and combining it with a standard long 250-μm ID first-dimension column. Compared with conventional GC × GC set-ups, an over 3-fold reduction of the analysis time was reached in the present study without sacrificing resolution; to the contrary, a substantial increase of the peak capacity was obtained

On-line coupling of immunoaffinity-based solid-phase extraction and gas chromatography for the determination of s-triazines in aqueous samples.

The potential of immunoaffinity-based solid-phase extraction (IASPE) coupled on-line to gas chromatography (GC) for the determination of micropollutants was studied with emphasis on the interfacing of the immunoaffinity-based SPE and GC parts of the system. The cartridge containing the immobilized antibodies was coupled to the gas chromatograph via a reversed-phase cartridge (copolymer sorbent). After trace enrichment of the analytes on the immunoaffinity cartridge, they were desorbed and recollected on the reversed-phase cartridge by means of an acidic buffer. After clean-up and drying with nitrogen, desorption and transfer to the GC was done with ethyl acetate via an on-column interface in the partially concurrent solvent evaporation mode. The antibodies used in the immunoaffinity cartridge were raised against atrazine; several s-triazines were used as test compounds. Triazines that were structurally similar to atrazine, showed quantitative recovery. As an application, immunoaffinity SPE-GC was used for the analysis of river and waste water and orange juice. The selectivity of the system was such that non-selective flame ionization detection (FID) could be used to detect the analytes of interest in these complex matrices. The detection limits for 10-ml water samples were 15-25 ng/l for FID and about 1.5 ng/l for the nitrogen-phosphorus detection. Copyright (C) 1999 Elsevier Science B.V

Determination of chlorophenoxy acid herbicides in water by in situ esterification followed by in-vial liquidBliquid extraction combined with large-volume on-column injection and gas chromatographyBmass spectrometry

A new approach for rapidly analysing chlorophenoxy acid herbicides in water is presented. The chlorinated acids are derivatised with dimethyl sulphate in the water sample itself (800 μl) and, next, the methyl esters are extracted with 800 μl of n-hexane. A 200-μl volume of the extract is injected into the GC-MS system. The miniaturisation of both the methylation and extraction steps could be implemented because of the use of large-volume on-column injection and mass spectrometric detection. The optimisation of the methylation reaction for the simultaneous determination of (3,6-dichloro-2- methoxy)benzoic acid, (2-methyl-4-chlorophenoxy)- and (2,4- dichlorophenoxy)acetic acids, (±)-2-(4-chloro-2-methylphenoxy)- and 2-(2,4- dichlorophenoxy)propanoic acids and 4-(4-chloro-2-methylphenoxy)- and 4-(2,4- dichlorophenoxy)butyric acids showed that tetrabutylammonium salts act as catalysts. Addition of sodium hydroxide was required to obtain quantitative reaction yields for 4-(4-chloro-2-methylphenoxy)- and 4-(2,4- dichlorophenoxy)butyric acids. The methylation-cum-extraction procedure takes only 3 min per sample for a batch of seven samples. Linear calibration plots were obtained for the complete procedure and the limits of detection were of 10-60 ng/l with a signal-to-noise ratio (S/N) of 6. Relative standard deviations ranged from 8 to 15% (n = 7) for analyte concentrations of 0.5 μg/l in surface water. (C) 2000 Elsevier Science B.V