In this work we analyzed the quaternary structure of FAD-dependent 3-ketosteroid dehydrogenase (AcmB) from Sterolibacterium denitrificans, the protein that in solution forms massive aggregates (>600 kDa). Using size-excursion chromatography (SEC), dynamic light scattering (DLS), native-PAGE and atomic force microscopy (AFM) we studied the nature of enzyme aggregation. Partial protein de-aggregation was facilitated by the presence of non-ionic detergent such as Tween 20 or by a high degree of protein dilution but not by addition of a reducing agent or an increase of ionic strength. De-aggregating influence of Tween 20 had no impact on either enzyme's specific activity or FAD reconstitution to recombinant AcmB. The joint experimental (DLS, isoelectric focusing) and theoretical investigations demonstrated gradual shift of enzyme's isoelectric point upon aggregation from 8.6 for a monomeric form to even 5.0. The AFM imaging on mica or highly oriented pyrolytic graphite (HOPG) surface enabled observation of individual protein monomers deposited from a highly diluted solution (0.2 μg/ml). Such approach revealed that native AcmB can indeed be monomeric. AFM imaging supported by theoretical random sequential adsorption (RSA) kinetics allowed estimation of distribution enzyme forms in the bulk solution: 5%, monomer, 11.4% dimer and 12% trimer. Finally, based on results of AFM as well as analysis of the surface of AcmB homology models we have observed that aggregation is most probably initiated by hydrophobic forces and then assisted by electrostatic attraction between negatively charged aggregates and positively charged monomers.
Keywords: Adsorption kinetics; Atomic force microscopy of proteins; Isoelectric point; Protein aggregation; Quaternary structure; Size excursion chromatography.
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