Structural Determinants of Cold Adaptation and Stability in a Large Protein

Abstract

The heat-labile α-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable α-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point Tm, the calorimetric enthalpy ΔHcal, the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters kcat and Km. The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins. Although all mutations were located far from the active site, their overall trend is to decrease both kcat and Km by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted α-amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation.