Computational protocol: Effect of Heavy Atoms on the Thermal Stability of α-Amylase from Aspergillus oryzae

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Protocol publication

[…] As a test protein for this work, we used commercially available α-amylase (endo-1,4-α-D-glucan glucohydrolase) from Aspergillus oryzae (EC, MW = 52.4×103, Sigma, Cat. No. 10065) without further purification. The optimal conditions for α-amylase in industrial application are pH and no Ca2+ , . The program HATODAS ( identified six candidate heavy atoms based on the amino-acid sequence of Ao α-amylase and pH 5.8 as the query terms. Ao α-amylase activity with the selected heavy atoms was examined using the starch-iodine assay method . A 0.1 mL aliquot of Ao α-amylase at 0.4 mg/mL in 0.1 M MES-NaOH (pH 5.8) with 1.0 mM heavy atoms was incubated for 5 min at 70°C in a water bath; this temperature was chosen because Td for Ao α-amylase is 61°C in the absence of heavy atoms. The reactions were initiated by adding 0.1 mL of 0.25% (w/v) starch solution as a substrate to α-amylase solutions at 20°C. The α-amylase activity was confirmed by adding 0.1 mL of iodine reagent [0.02% (w/v) I2, 0.2% (w/v) KI] to 0.2 mL of the protein-starch solution at 20°C. [...] Diffraction-quality crystals of Ao α-amylase were obtained using the oil microbatch method on the Autolabo automatic crystallization system . A crystallization drop of 1.0 µL, in the presence of synthetic zeolite molecular sieves as heteroepitaxic nucleants , , was created by mixing 1∶1 mixture of 28.0 mg/mL protein solution in 0.02 M MES-NaOH (pH 5.8) and precipitant solution composed of 40% (w/v) polyethylene glycol (PEG) 8,000, 0.2 M CaCl2, and 0.1 M MES-NaOH (pH 5.8) in a well of a Nunc HLA crystallization plate (Nalge Nunc International) which was then covered with 20 µL of paraffin oil. The resulting Ao α-amylase crystals were submitted to a heavy-atom derivatization experiment. The Gd derivative was prepared by soaking native crystals with 10 mM GdCl3, 40% (w/v) PEG 8,000, 0.2 M CaCl2, 0.1 M MES-NaOH (pH 5.8). Although the SmCl3 derivatization was attempted using concentrations in the range 0.1–10 mM, this treatment caused the crystals to crack, and structure determination was therefore impossible.All crystals were directly mounted in a cryoloop from the crystallization drop and flash-cooled at 100 K in a nitrogen gas stream. Complete diffraction data sets were collected using an in-house Rigaku R-AXIS VII image-plate detector with Cu Kα radiation and a Rigaku R-AXIS V image-plate detector with synchrotron radiation at BL26B1 of SPring-8, Japan . All data were processed using the program HKL-2000 . [...] Positioning of one Ao α-amylase molecule in the asymmetric unit was accomplished using the molecular-replacement method as implemented in the program MOLREP , based on the crystal structure deposited in the Protein Data Bank (PDB code 2taa). The structure of the Gd-bound protein was isomorphous to that of the native form and was determined by difference Fourier analysis using the corresponding model of the native structure. Manual model revision was performed using QUANTA2000 software (Accelrys Inc.).Bound ions were observed in the structure of the Gd-soaked crystal. Based on comparison of temperature factors to those of neighboring atoms, and from their coordination with ion-binding residues, the bound ions are most likely to be Gd ions from the heavy-atom soaking reagent. This interpretation is in agreement with the fact that strong signals are observed at the ion sites in the anomalous Fourier map at a wavelength of 1.000 Å, prepared using the program FFT in CCP4 suite .The program CNS was used for structure refinement and electron-density map calculation. Each cycle of refinement with bulk solvent and overall anisotropic B-factor corrections consisted of rigid-body refinement, simulated annealing incorporating the slow-cool protocol, positional refinement, and B-factor refinement (individual or group). Several cycles of model revision and refinement yielded the final models. The stereochemical quality of the final structures was verified using the program PROCHECK . Statistics of the data collection and refinement are shown in . The structural data are available in the Protein Data Bank under the accession numbers 3VX0, 3VX1. […]

Pipeline specifications

Software tools HATODAS, HKL-2000, Molrep, CCP4, CNS, PROCHECK
Application Protein structure analysis
Organisms Dipturus trachyderma, Aspergillus oryzae
Chemicals Gadolinium, Samarium