Anomalous Effect of Poly(ethylene)Glycol on Intermolecular Interaction and Protein Molecule Association

Abstract

Taka-amylase A (TAA) proteins purified from Aspergillus oryzae were investigated using dynamic and static light scattering for solutions containing poly(ethylene)glycol (PEG) with a molecular weight of 8000 as an association-inducing reagent. The hydrodynamic TAA monomer radius at a zero protein concentration gradually decreased from 3.2 to 2.0 nm with an increase in the PEG concentration from 0 to 12.5% (w/v). The molecular weight of TAA monomers at 0% PEG, 49.0 kDa, decreased to 31.0 kDa at 12.5% PEG. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis analysis showed that the monomer-band position decreased with an increase in the PEG concentration. While these findings suggest PEG-induced fragmentation of monomers, circular dichroism measurements showed no difference between 12.5 and 0% PEG solutions in the secondary structure of TAA monomers. Analytical ultracentrifugation with a 12.5% PEG solution gave a TAA molecular weight of 51.0 kDa, almost equal to that for a 0% PEG solution measured using static light scattering. These results suggest that a PEG-induced network locally formed in the solution and affected the dynamics and the scattering intensity of the TAA monomers. The existence of such a solution structure makes it impossible to explain the behavior of monomers on the basis of simple attractive depletion interaction by PEG.

Crystallization of Cellobiohydrolase in the Presence of Cellulose-Degraded Cellobiose: Analysis of Intermolecular Interactions and Association Dynamics

“…The association of cellobiohydrolase molecules after the protein solution (containing cellobiose) was mixed with the crystallization buffer at a volume ratio of 1:1 was observed using a multi-angle SLS detector [28][29][30][31][32]. The concentration of cellobiohydrolase in the mixed solution was 7 mg/mL and that of lithium sulfate was 1.25 M. The scattering intensities at q from 20˚ to 150˚ with a step of 1˚ were simultaneously measured over time at intervals of 3 s for each R(cb/ce) condition.…”

“…The Hamaker constant of cellobiohydrolase monomers in the standard buffer at each R(cb/ce) was estimated using the results of the intermolecular interaction parameter and surface charge [22][23][24]26,31,32]. The Derjaguin-Landau-Verwey-Overbeek model [33] was used to calculate the constant.…”

Relationship between molecular weight of poly(ethylene)glycol and intermolecular interaction of Taka-amylase A monomers

“…The purification and preparation of the TAA were carried out at 4 1C in the same manner as previously reported [27]. 2 g of TAA powder from Aspergillus oryzae (Sumizyme-L, Shin Nihon Chemical Co. Ltd.) were dissolved in a 30-mL solution containing 20 mM of HEPES-NaOH buffer with a pH of 7.0.…”

“…The protein solution was purified in a sequence of steps: anion exchange chromatography (Q-Sepharose FF column; GE Healthcare Bioscience), hydrophobic interaction chromatography (Phenyl ToyoPearl650s column; TOSO), and anion exchange chromatography (Q-Sepharose HP column; GE Healthcare Bioscience). The fractions corresponding to ''TAA-1'' [27] were collected, and their purities were analyzed using SDS and native PAGE. After the final chromatography, the purified solution was dialyzed with 50 mM of sodium acetate (NaAc)-acetic acid buffer and 2 mM of CaCl 2 at 20 1C with a pH of 6.0.…”

“…Our previous light-scattering investigation of Taka-amylase A (TAA), however, showed that this model cannot explain the behavior of TAA monomers in the PEG solution with a molecular weight of 8000 even though the solution condition is far from LLPS [21]. The present study was motivated by our finding that TAA single crystals barely grew in PEG 8000 solutions although they readily grow when inorganic salt is used.…”