Effect of microheterogeneity on horse spleen apoferritin crystallization

Apoferritin (APO) is an interesting model protein for crystal growth studies, as an alternative to the widely used hen egg white lysozyme. The effect of naturally occurring oligomers on the crystallization of isolated, microhomogeneous APO monomers (24 subunits, M-r = 440 000) was investigated. SDS PAGE analysis and immunoblotting showed that commercial APO was free of foreign proteins ( > 99.9% w/w). The quaternary structure of APO oligomers that form prior to the addition of precipitant was analyzed in native 4-15% T (1-2% C) gradient PAGE. Optical densitometry of these gels showed that oligomers ( > 24 subunit monomer) constituted approximately 45% w/w of the total APO. The primary oligomeric contaminants were dimers (48 subunits) with 35% w/w, and several bands constituting trimers (similar to 72 subunits) with 10% w/w. Directly determined physical molecular weights (M-w) and conformational data for oligomers obtained by analytical gel filtration fast protein-liquid-chromatography separations utilizing UV and multi-angle laser light scattering detectors (GF-FPLC-MALLS) confirmed and expanded the native PAGE results. This technique allowed the discovery of large oligomers (M-w = 5000000 and 80000000) present in concentrations <1% w/w. Semi-preparative GF-FPLC was used to quantitatively reduce oligomer contamination to 5% w/w, and to produce 0.25 g of microhomogeneous monomers from 0.5 g APO. Crystallization from microhomogeneous monomer solutions yielded large crystals 0.5-1.0 mm in size. These crystals yielded an X-ray diffraction resolution of 1.8 Angstrom. Reconstitutive experiments in which isolated oligomers were added to monomer preparations showed that dimers perturb the growth habit and reduce the crystal growth, without significantly affecting the nucleation. On trimer addition, the nucleation was increased and the crystal growth decreased. Addition of cadmium sulfate precipitant to unpurified APO did not affect the nature or quantity of the oligomers. These effects of oligomers on crystallization underline microheterogeneity as a critical factor in protein crystallization. (C) 1998 Elsevier Science B.V. All rights reserved.