EFFECT OF MICROHETEROGENEITY ON HORSE SPLEEN APOFERRITIN CRYSTALLIZATION

Apoferritin (APO) is an interesting model protein for crystal growth s tudies, as an alternative to the widely used hen egg white lysozyme. T he effect of naturally occurring oligomers on the crystallization of i solated, microhomogeneous APO monomers (24 subunits, M-r = 440 000) wa s investigated. SDS PAGE analysis and immunoblotting showed that comme rcial APO was free of foreign proteins ( > 99.9% w/w). The quaternary structure of APO oligomers that form prior to the addition of precipit ant was analyzed in native 4-15% T (1-2% C) gradient PAGE. Optical den sitometry of these gels showed that oligomers ( > 24 subunit monomer) constituted approximately 45% w/w of the total APO. The primary oligom eric contaminants were dimers (48 subunits) with 35% w/w, and several bands constituting trimers (similar to 72 subunits) with 10% w/w. Dire ctly determined physical molecular weights (M-w) and conformational da ta for oligomers obtained by analytical gel filtration fast protein-li quid-chromatography separations utilizing UV and multi-angle laser lig ht scattering detectors (GF-FPLC-MALLS) confirmed and expanded the nat ive PAGE results. This technique allowed the discovery of large oligom ers (M-w = 5000000 and 80000000) present in concentrations <1% w/w. Se mi-preparative GF-FPLC was used to quantitatively reduce oligomer cont amination to 5% w/w, and to produce 0.25 g of microhomogeneous monomer s from 0.5 g APO. Crystallization from microhomogeneous monomer soluti ons yielded large crystals 0.5-1.0 mm in size. These crystals yielded an X-ray diffraction resolution of 1.8 Angstrom. Reconstitutive experi ments in which isolated oligomers were added to monomer preparations s howed that dimers perturb the growth habit and reduce the crystal grow th, without significantly affecting the nucleation. On trimer addition , the nucleation was increased and the crystal growth decreased. Addit ion of cadmium sulfate precipitant to unpurified APO did not affect th e nature or quantity of the oligomers. These effects of oligomers on c rystallization underline microheterogeneity as a critical factor in pr otein crystallization. (C) 1998 Elsevier Science B.V. All rights reser ved.