Structural Determinants of Cold Adaptation and Stability in a Large Protein

Abstract

The heat-labile ␣-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 ␣-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point T m , the calorimetric enthalpy ⌬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 ␣-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.The cold-active and heat-labile ␣-amylase from the antarctic bacterium Pseudoalteromonas haloplanktis is an exceptional case because it is the largest known protein undergoing a reversible unfolding according to a two-state reaction pathway. It has been proposed that this unusual behavior is dictated by the requirement for an improved flexibility or plasticity of the protein molecule to perform catalysis at near-zero temperatures (1). Structural investigations have suggested that this is achieved by decreasing the number and strength of weak interactions stabilizing the native conformation (2-4). Accordingly, one can expect that any mutation designed to improve the stability of this fragile edifice will induce significant perturbations of the denaturation pattern, thereby giving evidence for the structural features linked to the unfolding parameters.Small globular proteins that unfold according to a pure twostate process, i.e. reversibly and without a stable intermediate between the native and the unfolded states, have allowed researchers to establish the thermodynamic stability function accounting for the Gibbs energy change associated with their denaturation over physiological and nonphysiological temperature ranges. The current availability of this thermodynamic function, mainly gained by differential scanning calorimetry, represents much more than the simple physicochemical characterization of a polymer because it offers a powerful tool for investigation of the still unexplained "second genetic code," i.e. the driving force allowing a polypeptide to fold into a predetermined native and biologically active conformation (5). The validity of the bell-shaped stabi...

Activity-Stability Relationships in Extremophilic Enzymes

“…These enzymes unfold at distinct denaturant concentrations (C1 ⁄2 ) and are characterized by a decrease of unfolding cooperativity (m value) and the appearance of intermediate states (BAA) as the stability increases. These observations parallel the behavior of protein adapted to different temperatures as recorded by thermal unfolding (12,13). Estimation of the conformational stability in the absence of denaturant (⌬G H 2 O ) at 20°C using Equation 4 provides a ratio of 1/2/6 for AHA, PPA, and BAA, respectively.…”

“…Sources-The recombinant AHA was expressed in Escherichia coli at 18°C and purified by DEAE-agarose, Sephadex G-100, and Ultrogel AcA54 column chromatography, as described previously (13). PPA and BAA were from Roche Molecular Biochemicals and Sigma, respectively.…”

“…These minima are responsible for the ruggedness of the funnel slopes and for the reduced cooperativity of the foldingunfolding reaction as demonstrated by GdmCl and heat-induced unfolding. In contrast, the structural elements of psychrophilic proteins generally unfold cooperatively without intermediates, as a result of fewer stabilizing interactions and stability domains (12,13), and therefore, the funnel slopes are steep and smooth. The bottom of the funnel, which depicts the stability of the native state ensemble, also displays significant differences between both extremozymes.…”

“…In the present work, we have investigated three structurally homologous ␣-amylases. The ␣-amylase from Pseudoalteromonas haloplanktis (AHA), 1 formerly Alteromonas haloplanctis, is the best characterized psychrophilic enzyme isolated from an Antarctic bacterium (12)(13)(14). Interestingly, its closest structural homologue is the pig pancreatic ␣-amylase (PPA), which is taken here as the mesophilic reference (15,16).…”

Stepwise Adaptations to Low Temperature as Revealed by Multiple Mutants of Psychrophilic α-Amylase from Antarctic Bacterium

“…To probe the activity-flexibility-stability hypothesis, numerous mutants of AHA have been constructed, each bearing additional weak interactions mediated by the replacement of a specific residue, as well as a disulfide bond as found in PPA. It has been shown that single amino acid side chain substitutions significantly modify the stability parameters, the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k cat and K m (25)(26)(27). Here, we report the activity and mainly the stability properties of two stabilized multiple mutants ( Fig.…”