Cold adaptation of proteins: a biophysical study of a psychrophilic alpha-amylase and its stabilized mutants

• Main Authors: Cipolla, Alexandre, D'Amico, Salvino, Feller, Georges
• Other Authors: CIP - Centre d'Ingénierie des Protéines - ULiège
• Format: Conference Object
• Language: English
• Published: 2010
• Subjects: thermal adaptation; folding; protein engineering; Life sciences; Biochemistry; biophysics & molecular biology; Sciences du vivant; Biochimie; biophysique & biologie moléculaire; Antarc*; Antarctic
• Online Access: https://orbi.uliege.be/handle/2268/93097

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 ...