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See detailAdaptations structurales et fonctionnelles aux températures extrêmes au sein de la famille des alpha-amylases chlorure-dépendantes
Cipolla, Alexandre ULg

Doctoral thesis (2012)

L’adaptation thermique des protéines extremophiles a été étudiée de manière à approfondir notre compréhension des mécanismes moléculaires qui en sont responsables. Dans ce but, deux études ont été ... [more ▼]

L’adaptation thermique des protéines extremophiles a été étudiée de manière à approfondir notre compréhension des mécanismes moléculaires qui en sont responsables. Dans ce but, deux études ont été initiées et publiées. La première est basée sur la "mésophilisation" de l’α-amylase psychrophile AHA, issue de la bactérie Antarctique Pseudoalteromonas haloplanktis. L’ajout d’interactions faibles et d’un pont disulfure présents chez son homologue mésophile PPA de Sus scrofa et absents chez AHA ont permis de construire deux mutants multiples stabilisés, Mut5 et Mut5CC. Ces quatre enzymes ont été étudiées sur base de leur stabilité, de leur activité et de la perméabilité de leur structure protéique. L’étude des cinétiques de renaturation/dénaturation d’AHA, Mut5 et Mut5CC a permis de déterminer l’origine cinétique du gain de stabilité liée à l’ajout d’interactions faibles et du pont disulfure chez AHA. Il en résulte que Mut5 et Mut5CC ont effectivement été stabilisés mais en contrepartie ils ont perdu l’optimalisation de l’activité à basse température observée chez AHA. De plus, la perméabilité de leur structure protéique s’est réduite, se rapprochant de celle de PPA. L’origine du gain de stabilité est liée à une diminution des cinétiques de dépliement sans modification des cinétiques de repliement. Non seulement ces résultats démontrent l’importance du rôle des interactions faibles dans l’adaptation thermique des protéines mais de plus, ils démontrent la synergie entre celles-ci. La seconde étude a pu être développée par la découverte d’une α-amylase chlorure-dépendante thermophile TFA issue de l’actinomycète Thermobifida fusca et par la production de l’α-amylase chlorure-dépendante mésophile ectotherme DMA de Drosophila melanogaster. Ainsi avec AHA et PPA respectivement comme représentants psychrophile et mésophile homéotherme, nous pouvions couvrir l’ensemble des températures physiologiques/environnementales connues. Nous avons pu mettre en évidence le continuum des propriétés physico-chimiques observées (activité, stabilité, affinité pour le substrat…) mais aussi que l’énergie thermique de l’environnement influence grandement l’activité enzymatique qui ne serait pas contrebalancée par les mécanismes adaptatifs. L’influence de la température sur l’activité a mis en évidence la plus faible dépendance d’AHA par rapport à TFA. Ces travaux ont permis d’améliorer notre compréhension des mécanismes moléculaires liés à l’adaptation thermique des protéines et du rôle joué par les interactions faibles dans cette adaptation. Ils ouvrent aussi la voie à de futures recherches visant à analyser par d’autres méthodes la flexibilité de la structure protéique et à cristalliser TFA pour étudier d’un point de vue structural le continuum des propriétés physico-chimiques mis en évidence au cours de ce travail. [less ▲]

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See detailLife in the cold: proteomics of the Antarctic bacterium Pseudoalteromonas haloplanktis
Piette, Florence ULg; Struvay, Caroline ULg; Godin, Amandine ULg et al

in Heazlewood, J. L.; Petzold, C. J. (Eds.) Proteomic Applications in Biology (2012)

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See detailActivity-Flexibility and Stability Relationships as revealed by multiple mutants of a psychrophilic alpha-Amylase
Cipolla, Alexandre ULg; D'Amico, Salvino ULg; Feller, Georges ULg

Poster (2011, May 23)

Permanently cold environments, like polar regions, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently at low temperatures. We have ... [more ▼]

Permanently cold environments, like polar regions, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently at low temperatures. We have investigated the role of weak interactions in thermal adaptation of proteins by site-directed mutagenesis of the psychrophilc alpha-amylase (AHA) from the Antarctic bacterium Pseudoalteromonas haloplanktis. Two stabilized multiple-mutants (Mut5 and Mut5CC) have been constructed. The single mutations were selected by comparison of the presence of weak interactions in a mesophilic homolog from pig pancreas, PPA. The three enzymes AHA, Mut5 and Mut5CC have been analyzed by differential scanning calorimetry, thermal and chemical denaturation. The flexibility has been studied by acrylamide-induced fluorescence quenching. In order to investigate the kinetic origin of the gain in stability, the kinetics of unfolding and refolding in GdmCl have been monitored at 15°C. The newly introduced weak interactions stabilized the mutants, protected them against heat and chemical unfolding and also induced an effective loss of flexibility. In addition, the two multiple-mutants exhibit an increased optimum temperature for activity. The first results of kinetic studies show a similar refolding phase but differences between the three amylases in the unfolding phase. These results unambiguously support the capital role of weak interactions in the balance between activity, flexibility and stability and provide a better knowledge of the adaptation of enzymes to cold temperatures. [less ▲]

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See detailStepwise adaptations to low temperature as revealed by multiple mutants of a psychrophilic alpha-amylase from an Antarctic bacterium
Cipolla, Alexandre ULg; D'Amico, Salvino ULg; Barumandzadeh, Roya et al

in Journal of Biological Chemistry (2011), 286(44), 3834838355

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See detailPsychrophilic enzymes: cool responses to chilly problems
Roulling, Frédéric ULg; Piette, Florence ULg; Cipolla, Alexandre ULg et al

in Horikoshi, K.; Antranikian, G.; Bull, A. (Eds.) et al Extremophiles Handbook (2011)

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See detailCold adaptation of proteins: a biophysical study of a psychrophilic alpha-amylase and its stabilized mutants
Cipolla, Alexandre ULg; D'Amico, Salvino ULg; Feller, Georges ULg

Poster (2010, September 28)

Habitats of permanently cold temperature, like polar regions for example, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently in these cold ... [more ▼]

Habitats of permanently cold temperature, like polar regions for example, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently in these cold environments. According to the hypothesis developed in our laboratory, the adaptation to cold temperature involves relationships between activity, flexibility and stability. Even if activity and stability are not physically linked in proteins 1, the consensus for the adaptive strategy is to take advantage of the lack of selective pressure for stable proteins to lose stability, therefore increasing the flexibility or mobility of the enzyme at low temperatures that restrict molecular motions. 2 Working on alpha-amylase, we have investigated the role of weak interactions in thermal adaptation of proteins by site-directed mutagenesis. We have built two multiple-mutants (Mut5 and Mut5CC) of the psychrophilc alpha-amylase (AHA) from the Antarctic bacterium, Pseudoalteromonas haloplanktis. The single mutations were selected by comparison of the presence of weak interactions in a mesophilic chloride-dependant homolog from pig pancreas, PPA. The study of selected single mutations prompt us to construct two multiple-mutants, Mut5 and Mut5CC, carrying 5 and 6 additional weak interactions found in PPA, that showed an increased stability and a lower activity at 25 °C.3 We have compared AHA, Mut5 and Mut5CC with additional methods like differential scanning calorimetry, thermal and chemical unfolding in order to determine the gain in stability. We also studied the flexibility or breathing of the enzymes by acrylamide-induced fluorescence quenching and we determined the optimum activity temperature for the three amylases. In order to investigate the kinetic origin of the gain in stability 4 for the two multiple-mutants, we studied in a first step the kinetic unfolding and refolding by GdmCl of the three amylases by manual methods following fluorescence signal at 15°C. The newly introduced weak interactions stabilized the proteins, protected them against heat and chemical unfolding and also induced an effective loss of flexibility. In addition, the two multiple-mutants exhibit a different optimum activity temperature than AHA. The first result in manual kinetic studies seems to show a similar refolding phase but a difference between the three amylases in the unfolding phase. This is in correlation with results of Dieter, P et al 4. These results and those of the previous work 3, unambiguously support the capital role of weak interactions in the balance between activity, flexibility and stability and provide a better knowledge of the adaptation of enzymes to cold temperatures. [less ▲]

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See detailTemperature Adaptation of Proteins: Stability, Folding and Flexibility in Mesophilic-like Engineered Alpha-Amylases
Cipolla, Alexandre ULg; D'Amico, Salvino ULg; Feller, Georges ULg

Poster (2009, July 02)

Habitats of permanently cold temperature, like polar regions for example, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently in these cold ... [more ▼]

Habitats of permanently cold temperature, like polar regions for example, have been colonized by a great variety of psychrophilic organisms producing enzymes adapted to function efficiently in these cold environments. According to the hypothesis developed in our laboratory, the adaptation to cold temperature involves relationships between activity, flexibility and stability. Even if activity and stability are not physically linked in proteins 1, the consensus for the adaptive strategy is to take advantage of the lack of selective pressure for stable proteins to lose stability, therefore increasing the flexibility or mobility of the enzyme at low temperatures that restrict molecular motions. 2 Working on alpha-amylase, we have investigated the role of weak interactions in thermal adaptation of proteins by site-directed mutagenesis. We have built two multiple-mutants (Mut5 and Mut5CC) of the psychrophilc alpha-amylase (AHA) from the Antarctic bacterium, Pseudoalteromonas haloplanktis. The single mutations were selected by comparison of the presence of weak interactions in a mesophilic chloride-dependant homolog from pig pancreas, PPA. The study of selected single mutations prompt us to construct two multiple-mutants, Mut5 and Mut5CC, carrying 5 and 6 additional weak interactions found in PPA, that showed an increased stability and a lower activity at 25 °C.3 We have compared AHA, Mut5 and Mut5CC with additional methods like differential scanning calorimetry, thermal and chemical unfolding and circular dichroism in order to determine the gain in stability. We also studied the flexibility or breathing of the enzymes by acrylamide-induced fluorescence quenching. The newly introduced weak interactions stabilized the proteins, protected them against heat and chemical unfolding and also induced an effective loss of flexibility. These results and those of the previous work 3, unambiguously support the capital role of weak interactions in the balance between activity, flexibility and stability and provide a better knowledge of the adaptation of enzymes to cold temperatures. [less ▲]

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