Effect of heavy atoms on the thermal stability of α-amylase from Aspergillus oryzae.

Currently, there are no versatile and established methods for improving stability of proteins. In an entirely different approach from conventional techniques such as mutagenesis, we attempted to enhance enzyme stability of α-amylase from Aspergillus oryzae using a heavy-atom derivatization technique. We evaluated changes in stability using differential scanning calorimetry (DSC). Candidate heavy atoms were identified using the Heavy-Atom Database System HATODAS, a Web-based tool designed to assist in heavy-atom derivatization of proteins for X-ray crystallography. The denaturation temperature of α-amylase derivatized with gadolinium (Gd) or samarium (Sm) ions increased by 6.2 or 5.7°C, respectively, compared to that of the native protein (60.6°C). The binding of six Gd ions was confirmed by X-ray crystallography of the enzyme at 1.5 Å resolution. DSC and dynamic light-scattering data revealed a correlation between stability and the aggregation state upon addition of Gd ions. These results show that HATODAS search is an effective tool for selecting heavy atoms for stabilization of this protein

The crystal structure of Gd-derivatized Ao α-amylase.

<p>(A) Overall structure shown in ribbon diagram. The three domains, A, B, and C, are colored light blue, brown, and orange, respectively. NAG molecule and residue Asn197 are depicted as licorice models. Bound Ca and Gd ions are depicted as pink and yellow spheres, respectively. Close-up view of (B) NAG binding site with (2<i>F</i>o–<i>F</i>c) electron-density map contoured at 1.2<i>σ</i> (blue mesh) and 0.6<i>σ</i> (orange mesh) level and (C) Ca binding site. The residues are depicted as licorice models. The perspective is the same as that in Fig. 2A. Drawn in <i>QUANTA2000</i>.</p

Data-collection and refinement statistics.

<p>Values in parentheses are for the outermost shell.</p>†<p><i>R</i>_merge = ∑<i>_hkl</i> ∑<i>_i</i> |<i>I_i</i>(<i>hkl</i>)−<<i>I</i>(<i>hkl</i>)>|/∑<i>_hkl</i> ∑<i>_i I_i</i>(<i>hkl</i>), where <i>I_i</i>(<i>hkl</i>) is the <i>i</i>th observation of reflection <i>hkl</i> and <<i>I</i>(<i>hkl</i>)> is the weighted average intensity for all observations <i>i</i> of reflection <i>hkl</i>.</p>§<p><i>R</i>_cryst = ∑<i>_hkl</i> ||<i>F</i>_obs|−|<i>F</i>_calc||/∑<i>_hkl</i> |<i>F</i>_obs|, where |<i>F</i>_obs| and |<i>F</i>_calc| are the observed and calculated structure-factor amplitudes, respectively. <i>R</i>_free was calculated with 5% of the reflections chosen at random and omitted from refinement.</p

Results of DSC and DLS experiments for 0.4 mg/mL Ao α-amylase in the presence of Gd ions at pH 5.8.

*<p>molecular mass estimated from the measured radius (<i>DYNAMICS</i>, Protein Solutions).</p><p>A maximum <i>T</i>_d is observed at a concentration of 0.1 m<i>M</i> GdCl₃ (bold).</p

Close-up view of Gd binding sites.

<p>(A–E) Bound Gd ions and the interacting residues are depicted as yellow spheres and licorice models, respectively. The neighboring symmetry-related chains of domain A and B are colored blue and purple, respectively. The perspective is the same as that in Fig. 2A. Drawn in <i>QUANTA2000</i>.</p

DSC curves of Ao α-amylase with heavy atoms at pH 5.8.

<p>(A) 0.2 mg/mL Ao α-amylase with heavy atoms. (I) no heavy atom, (II) 0.2 m<i>M</i> GdCl₃, (III) 0.2 m<i>M</i> SmCl₃. (B) Gd concentration dependence of the denaturation temperature of 0.4 mg/mL Ao α-amylase with 0–2.0 m<i>M</i> GdCl₃ (0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.15, 0.2, 0.3, 0.5, 0.9, 1.0, and 2.0 m<i>M</i>). A concentration of 0.1 m<i>M</i> GdCl₃ gave the highest <i>T</i>_d value, 67.3°C (heavy line). The denaturation temperature, <i>T</i>_d, represents the temperature corresponding to the peak of the DSC curve observed at a scan rate of 200°C/hour.</p