ORBi: Cipolla Alexandre - Activity-Flexibility and Stability Relationships as revealed by multiple mutants of a psychrophilic alpha-Amylase

Reference : Activity-Flexibility and Stability Relationships as revealed by multiple mutants of a ps...


	Document type :	Scientific congresses and symposiums : Poster
	Discipline(s) :	Life sciences : Biochemistry, biophysics & molecular biology
	To cite this reference:	http://hdl.handle.net/2268/93098

Title :	Activity-Flexibility and Stability Relationships as revealed by multiple mutants of a psychrophilic alpha-Amylase
Language :	English
Author, co-author :	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 >]
Publication date :	23-May-2011
Page number :	A0
On invitation :	No
Audience :	International
Event name :	IX European symposium of The Protein Society: Wonders and disasters of the protein world
Event date :	du 22 Mai 2011 au 26 Mai 2011
Event organizer :	Stockholm University
	The Protein Society
Event place (city) :	Stockholm
Event country :	Suede
Keywords :	[en] thermal adaptation ; folding ; protein engineering
Abstract :	[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.
Research units :	Centre d'Ingénierie des Protéines - CIP
Funders :	Fonds de la Recherche Scientifique (Communauté française de Belgique) - F.R.S.-FNRS FRIA ; Patrimoine Université de Liège
Name of the research project :	Identification des facteurs structuraux responsables de l’activité aux basses températures chez une -amylase produite par une bactérie psychrophile de l’Antarctique
Target :	Researchers
Permalink :	http://hdl.handle.net/2268/93098

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	File	Commentary	Version	Size	Access
Restricted access	Cold adaptation of proteins.pdf	Poster	Author postprint	1.1 MB	Request copy
Restricted access	ACR44839.4720E.pdf	Abstract	Publisher postprint	114.55 kB	Request copy

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