Reference : Activity-Flexibility and Stability Relationships as revealed by multiple mutants of a...
Scientific congresses and symposiums : Poster
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/93098
Activity-Flexibility and Stability Relationships as revealed by multiple mutants of a psychrophilic alpha-Amylase
English
Cipolla, Alexandre mailto [Université de Liège - ULg > Département des sciences de la vie > Labo de biochimie >]
D'Amico, Salvino [Université de Liège - ULg > > GIGA-Research >]
Feller, Georges mailto [Université de Liège - ULg > Département des sciences de la vie > Labo de biochimie >]
23-May-2011
A0
No
No
International
IX European symposium of The Protein Society: Wonders and disasters of the protein world
du 22 Mai 2011 au 26 Mai 2011
Stockholm University
The Protein Society
Stockholm
Suede
[en] thermal adaptation ; folding ; protein engineering
[en] 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.
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
Researchers
http://hdl.handle.net/2268/93098

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