Reference : Cold adaptation of proteins: a biophysical study of a psychrophilic alpha-amylase and...
Scientific congresses and symposiums : Poster
Life sciences : Biochemistry, biophysics & molecular biology
Cold adaptation of proteins: a biophysical study of a psychrophilic alpha-amylase and its stabilized mutants
Cipolla, Alexandre mailto [Université de Liège - ULiège > Département des sciences de la vie > Labo de biochimie >]
D'Amico, Salvino [Université de Liège - ULiège > > GIGA-Research >]
Feller, Georges mailto [Université de Liège - ULiège > Département des sciences de la vie > Labo de biochimie >]
OzBio2010: The Molecules of Life - From Discovery to Biotechnology
du 26 septembre au 1er octobre 2010
University of Melbourne
[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 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.
Centre d'Ingénierie des Protéines - CIP
Fonds de la Recherche Scientifique (Communauté française de Belgique) - F.R.S.-FNRS - FRIA ; Patrimoine Université de Liège
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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