The Cold-Active M1 Aminopeptidase from the Arctic Bacterium Colwellia psychrerythraea

in Rawlings, Neil D.; Salvesen, Guy S. (Eds.) Handbook of Proteolytic Enzymes (Third Ed) (2013)

Life in the cold: proteomics of the Antarctic bacterium Pseudoalteromonas haloplanktis

in Heazlewood, J. L.; Petzold, C. J. (Eds.) Proteomic Applications in Biology (2012)

Temperature adaptations in psychrophilic, mesophilic and thermophilic chloride-dependent alpha-amylases

in Biochimie (2012), 94

Decoding the folding of Burkholderia glumae lipase: folding intermediates en route to kinetic stability

in PLoS ONE (2012), 7(5), 36999

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

Poster (2011, May 23)

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 ... [more ▼]

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. [less ▲]

Cytoplasmic and Periplasmic Proteomic Signatures of Exponentially Growing Cells of the Psychrophilic Bacterium Pseudoalteromonas haloplanktis TAC125

in Applied & Environmental Microbiology (2011), 77(4), 1276-1283

The protein folding challenge in psychrophiles: facts and current issues

in Environmental Microbiology (2011), 13

Stepwise adaptations to low temperature as revealed by multiple mutants of a psychrophilic alpha-amylase from an Antarctic bacterium

in Journal of Biological Chemistry (2011), 286(44), 3834838355

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

Poster (2010, September 28)

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 ... [more ▼]

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. [less ▲]

Protein stability and enzyme activity at extreme biological temperatures

in Journal of Physics : Condensed Matter (2010), 22