Health consciousness drives people to drink skim milk. Yet, improving the blue-white appearance and watery texture of skim milk is necessary to make consumers happy with skim milk. In this study, the influence of limited proteolysis with soluble or immobilized proteases, heat treatment, and pH on the whiteness of skim milk were examined to meet this goal. Limited proteolysis with milk-clotting enzymes increased the whiteness of skim milk. the proteases porcine pepsin and chymosin were immobilized onto nonporous ceramic, glass, and controlled pore glass (CPG) beads. The enzymes were coupled to beads either directly or via crosslinker proteins. Pepsin, immobilized onto CPG beads via crosslinker proteins, exhibited the best properties with respect to enzymatic activity, stability, and whitening efficiency. The L value (whiteness) of this immobilized enzyme-treated skim milk was 79.5, which approached the whiteness of 1% fat milk. Immobilized proteases whitened skim milk more effectively than did soluble proteases. The whiteness of skim milk was determined at various temperatures from 4 to 90°C. The L value increased with increasing temperature throughout the range tested. For samples not heated above 50°C, the increases in L values were completely reversible on cooling. Partial reversibility was observed with samples heated at 70°C and above. Milk whiteness was also determined at different pH values ranging from 5.0 to 8.2 at temperatures of 4, 20, and 30°C. The L value increased with decreasing pH and increasing temperature. A maximum L value of 80.0 was obtained at pH 5.0 and 30°C, which is higher than the L value of skim milk at its natural pH at room temperature. The temperature-dependent dissociation of major caseins was investigated by size exclusion chromatography at temperatures from 10 to 40°C. Free soluble β-casein and κ-casein were found only at 10°C.

Alzheimer’s disease is the most common cause of dementia and was responsible for over 2% of all deaths in Sweden 2012. One of the pathological hallmarks is amyloid plaques built by fibrillated Amyloid β. Luminescent conjugated oligothiophenes are known to stain and give characteristic fluorescence spectra when staining amyloid fibrils. Little is however known about the interactions between LCOs and fibrils. Studies have been performed on molecules more traditionally known to stain amyloid fibrils. Studies have also been performed on fibrils using limited proteolysis. So far no studies have been performed using LCOs combined with limited proteolysis in order to study the interaction pattern between LCOs and fibrils. Amyloid β is expressed and purified using a simple few step purification protocol. The amyloid β peptide was then fibrillated in several generations in order to select for a homogenous fibril structure. This purification protocol also has the ability to purify different oligomers of Amyloid β that are interesting from a toxicity point of view. In this thesis optical characteristics and limited proteolysis with mass spectrometry are being used to studies the interactions between LCOs and fibrillated amyloid β. The proteolytic pattern was suggestive of an accessible N-terminal and a hidden C-terminal of Amyloid β M1-42 in the fibril. It was also shown that the proteolysis cleavage pattern of Chymotrypsin is not disrupted when the LCO pKTAA was used to stain fibrils. The emission spectra from the two LCOs pATAA and pKTAA changes differently when subjected to continuous excitation indicative of conformational changes or chemical modification.

It is well-known that the large diversity of protein functions and structures derives from the broad spectrum of physicochemical properties of the 20 canonical amino acids that constitute modern proteins. According to the generally accepted coevolution theory of the genetic code, evolution of protein structures and functions was continuously associated with enrichment of the genetic code, with aromatic amino acids being considered the latest addition to the genetic code to increase structural stability of proteins and diversification of their catalytic functions. The main objective of this master thesis was to test whether enzymatic catalysis could precede the appearance of aromatic amino acids in the standard genetic code. For that purpose, the effect of amino acid alphabet reduction on structure and function of dephosphocoenzyme A kinase (DPCK) was studied. Dephosphocoenzyme A kinase catalyses the final step in the biosynthesis of coenzyme A, a very conserved cofactor. Two aromatic amino acid-lacking mutants of DPCK from a thermophilic bacterium, Aquifex aeolicus, were designed by substituting aromatic amino acid residues by (i) leucines and (ii) various non-aromatic amino acids to best preserve the structural stability of the protein. Wild type protein and the two mutants were cloned and...

The Calvin – Benson cycle is the most widespread metabolic pathway capable of fixing CO2 in nature and a target of very high interest to metabolic engineers worldwide. In this study, 12 metabolites (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) were tested for protein – metabolite interactions against the proteome of Cupriavidus necator (previously Ralstonia eutropha) in the hopes of finding potential examples of allosteric regulation of the Calvin – Benson cycle. This is accomplished through the use of the LiP-SMap method, a recently developed shotgun proteomics method described by Piazza et al. capable of testing a metabolite of interest for interactions with the entire proteome of an organism at once. A functional protocol was developed and 234 protein – metabolite interactions between ATP and the proteome of C. necator are identified, 103 of which are potentially novel. Due to time constraints and setbacks in the lab, significant results were not produced for the other 11 metabolites tested. C. necator is an industrially relevant chemolithoautotroph that can be engineered to produce many valuable products and is capable of growth on CO2 and hydrogen gas. The bacteria were grown in continuous cultures after which the proteome was extracted while retaining its native state. Subsequently, the proteome was incubated with a metabolite of interest and subjected to limited, non-specific proteolysis. The resulting peptide mix was analyzed by liquid chromatography coupled tandem mass spectrometry (LC – MS/MS). / Calvin-Benson-cykeln är den mest utbredda metaboliska processen i naturen med vilken det är möjligt att fixera CO2 och en måltavla av högsta intresse för bioteknologer världen över. I den här studien testades 12 metaboliter (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) för interaktioner mot proteomet från Cupriavidus necator (tidigare Ralstonia eutropha) i hopp om att hitta potentiella exempel på allosterisk reglering av Calvin-Benson-cykeln. Detta uppnåddes genom användning av LiP-SMap-metoden, en nyligen utvecklad proteomikmetod beskriven av Piazza et al. kapabel av att testa en metabolit av intresse mot en organisms hela proteom simultant. Ett funktionellt protokoll utvecklades och 234 interaktioner mellan ATP och proteomet av C. necator identifierades, varav 103 potentiellt är nyupptäckta. På grund av tidsbrist och motgångar i labbet producerades inga signifikanta resultat för de resterande 11 metaboliterna som testades. C. necator är en industriellt relevant kemolitoautotrof som kan växa på CO2 och vätgas, samt manipuleras till att producera många värdefulla produkter. Bakterierna odlades i kemostater varefter proteomet extraherades i sitt naturliga tillstånd. Sedan inkuberades proteomet med en metabolit av intresse och utsattes för begränsad, icke-specifik proteolys. Den resulterande peptidblandningen analyserades via tandem masspektrometri kopplad till vätskekromatografi (LC – MS/MS).

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De nombreuses fonctions cellulaires essentielles telles que la traduction, l'épissage, la biogenèse des ribosomes et la réplication des télomères font appels aux particules RNP non codantes. La biogenèse de ces dernières chez les eucaryotes est un processus très complexes qui fait intervenir de nombreux facteurs cellulaires. La biogenèse du ribosome nécessite au moins 150 facteurs. Ceux-ci sont importants pour faciliter mais également contrôler la biogenèse de cette machinerie cellulaire essentielle qu'est le ribosome. Parmi ces facteurs, nous avons les snoRNP à boîtes C/D. Ces RNP sont impliqués dans la maturation des pré-ARNr (méthylation post-transcriptionnelle des riboses et clivages endonucléolytiques). Récemment notre laboratoire a participé à la découverte de facteurs d'assemblage de ces RNP. Il s'agit entre autre des protéines Rsa1p, du complexe R2TP (Rvb1p, Rvb2p, Tah1p et Pih1p) et de Hit1p chez la levure Saccharomyces cerevisiae. En utilisant une approche de co-expression à haut débit, nos travaux ont révélé un réseau complexe d'interactions entre les protéines constitutives des snoRNP et leurs facteurs d'assemblage. Couplé à une stratégie de protéolyse ménagée, la co-expression nous a permis d'obtenir des sous-complexes protéiques Snu13p/Rsa1p et Rsa1p/Hit1p qui font actuellement l'objet d'une étude structurale par RMN. En collaboration avec l'équipe de F. Allain (ETH Zurich), nous avons également déterminé la structure tridimensionnelle de la protéine Tah1p, ainsi que de son complexe avec le peptide C-terminale de la protéine chaperonne Hsp90 à haute résolution. Ces travaux ont révélé un mode d'association particulier entre le domaine TPR de la protéine et le peptide / A lot of essential cellular functions like translation, splicing, ribosome biogenesis and telomere replication need the activity of non coding RNPs. The biogenesis of non coding RNPs in eukaryotes is a complex pathway involving numerous cellular factors. For instance, ribosome biogenesis requires more than 150 factors. They are important to facilitate and to control the biogenesis of this essential cellular machinery. These factors include the C/D box snoRNPs. These RNPs are involved in pre-rRNA maturation (post-transcriptional ribose methylation and endo-nucleolytic cleavages). Recently, our laboratory participated to the discovery of snoRNP assembly factors: the Rsa1p protein, R2TP complex (Rvb1p, Rvb2p, Tah1p and Pih1p) and Hit1p in the yeast Saccharomyces cerevisiae. Using a high throughput co-expression approach, we deciphered a network of interactions between RNP core proteins and the assembly factors. Coupled with a limited proteolysis strategy, the co-expression method allowed us to obtain proteins sub-complexes Snu13p/Rsa1p and Rsa1p/Hit1p which are currently studied by NMR. In collaboration with the F. Allain team (ETH Zurich), we also determined the tridimensional structure of protein Tah1p and its complex with the chaperon Hsp90 C-terminal peptide at high resolution. The data obtained reveal a particular mode of association of the Tah1p TPR domain with the Hsp90 peptide

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Les dépôts intracellulaires de la protéine Tau agrégée sont la caractéristique commune des tauopathies, une famille de maladies neurodégénératives dont fait partie la maladie d’Alzheimer. Alors que les isoformes de Tau contenant trois (3R) ou quatre (4R) domaines de liaison aux microtubules sont retrouvées à des niveaux similaires dans le cerveau des individus sains, elles diffèrent au sein des inclusions intracellulaires en fonction des tauopathies. Notre étude repose sur l’identification de déterminants structuraux communs et distincts de fibres de Tau 3R et 4R. Pour cela deux approches de protéomique structurale complémentaires ont été mises au point à partir de fibres de Tau 1N3R et 1N4R produites in vitro. La première stratégie, reposant sur l’utilisation de protéolyses ménagées, nous a permis d’identifier les fragments protéolytiques qui composent un « code-barre » moléculaire propre à chaque assemblage. La seconde stratégie a utilisé un marquage chimique covalent des lysines accessibles suivi de l’analyse qualitative et quantitative des acides aminés marqués par spectrométrie de masse. Nous avons ainsi pu montrer que la partie N-terminale de la protéine était accessible au sein des fibres 1N3R et 1N4R tandis que la région C-terminale de la protéine est protégée pour Tau 1N3R et accessible au solvant pour Tau 1N4R. Nos résultats ouvrent la voie à de nouveaux outils moléculaires pour discriminer les tauopathies. / Intracellular deposits of Tau protein aggregates are the common hallmark of tauopathies, a range of neurodegenerative diseases including Alzheimer's disease. Levels of tau with three (3R) or four (4R) microtubule binding repeats are found similar in the normal adult brain, whereas they differ in neuropathological intracellular Tau inclusions, according to the type of tauopathy. Our study consists of the identification of common and different structural molecular determinants of 3R and 4R Tau fibrils. To this end, two proteomic approaches were optimized using 1N3R and 1N4R recombinant fibrils. The first strategy, using limited proteolysis, allowed us to identify the proteolytic fragments composing the molecular “bar-code” for each type of fibril. The second strategy we optimized used chemical covalent surface labelling of accessible lysines, and qualitative and quantitative analysis of the biotinylated residues using mass spectrometry. We show that, while the N-terminal part of the protein remains accessible within 1N3R and 1N4R fibrils, the C-terminal region is protected within 1N3R yet solvent accessible for 1N4R assemblies. Our results pave the way to new molecular tools to discriminate tauopathies.

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