Structural determinants of cold adaptation and stability in a large protein

The Journal of Biological Chemistry
S D'AmicoG Feller

Abstract

The heat-labile alpha-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 alpha-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point T(m), the calorimetric enthalpy Delta H(cal), the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k(cat) and K(m). 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 k(cat) and K(m) by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted alpha-amylase has lost a large number of weak interactions during evolution to reach the required conformational plas...Continue Reading

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