Stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein.

The stability of Rhodobacter capsulatus bacterioferritin, a 24-meric homopolymer, toward denaturation on variation in pH and temperature, and increasing concentrations of urea and guanidine.HCl was investigated with native PAGE, and CD and fluorescence spectroscopies. With temperature and urea, the wild-type protein denatured without discernible intermediates in the equilibrium experiments, but with guanidine.HCl (Gnd.HCl) one or more intermediate species were apparent at relatively low Gnd.HCl concentrations. Dissociated subunit monomers, or aggregates smaller than 24-mers containing the high alpha-helical content characteristic of the native protein were not obtained at any pH without a high proportion of the 24-mer being present, and taken together with the other denaturation experiments and the construction of stable subunit dimers by site-directed mutagenesis, this observation indicates that folding of the bacterioferritin monomer could be coupled to its association into a dimer. Glu 128 and Glu 135 were replaced by alanine and arginine in a series of mutants to determine their role in stabilizing the 24-meric oligomer. The Glu128Ala, Glu135Ala and Glu135Arg variants retained a 24-meric structure, but the Glu128Ala/Glu135Ala and Glu128Arg/Glu135Arg variants were stable subunit dimers. CD spectra of the Glu135Arg, Glu128Ala/Glu135Ala, and Glu128Arg/Glu135Arg variants showed that they retained the high alpha-helical content of the wild-type protein. The 24-meric Glu135Arg variant was less stable than the wild-type protein (T(m), [Urea](50%) and [Gnd.HCl](50%) of 59 degrees C, 4.9 M and 3.2 M compared with 73 degrees C, approximately 8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T(m), [Urea](50%) and [Gnd.HCl](50%) of 43 degrees C, approximately 3.2 M and 1.8 M, respectively). The differences in stability are roughly additive, indicating that the salt-bridges formed by Glu 128 and Glu 135 in the native oligomer, with Arg 61 and the amino-terminal amine of neighboring subunits, respectively, contribute equally to the stability of the subunit assembly. The additivity and assembly states of the variant proteins suggest that the interactions involving Glu 128 and Glu 135 contribute significantly to stabilizing the 24-mer relative to the subunit dimer.