|Reference : Temperature Adaptation of Proteins: Stability, Folding and Flexibility in Mesophilic-lik...|
|Scientific congresses and symposiums : Poster|
|Life sciences : Biochemistry, biophysics & molecular biology|
|Temperature Adaptation of Proteins: Stability, Folding and Flexibility in Mesophilic-like Engineered Alpha-Amylases|
|Cipolla, Alexandre [Université de Liège - ULg > Département des sciences de la vie > Labo de biochimie >]|
|D'Amico, Salvino [Université de Liège - ULg > > GIGA-Research >]|
|Feller, Georges [Université de Liège - ULg > Département des sciences de la vie > Labo de biochimie >]|
|Symposium in honour of Prof. Jean-Marie Frère "Penicillin-recognizing enzymes: from enzyme kinetics to protein folding"|
|du 01 juillet 2009 au 3 juillet 2009|
|Centre for Protein Engineering, University of Liège, Belgium|
|[en] thermal adaptation ; folding ; protein engineering|
|[en] 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.
|Centre d'Ingénierie des Protéines - CIP|
|Fonds de la Recherche Scientifique (Communauté française de Belgique) - F.R.S.-FNRS FRIA|
|Identification des facteurs structuraux responsables de l’activité aux basses températures chez une -amylase produite par une bactérie psychrophile de l’Antarctique|
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