The thermal stability of fibritin from bacteriophage T4 and its truncated mutants

The thermal stability of a series of recently obtained mutants of fibritin from bacteriophage T4 (a superhelical fibrous homotrimer with parallel-pack ed subunits each containing 486 amino acid residues) progressively truncate d from the subunit N-end was studied during incubation at 40-90 degrees C i n the presence of a surfactant (2% SDS). The mutant fibritins, G, B, C, and E, contained 443, 276, 231, and 120 amino acid residues, respectively. One more truncated mutant (fibritin S1, 108 amino acid residues) was obtained. The 2% SDS-PAGE showed that the migration mobilities of all these proteins corresponded to apparent molecular masses substantially greater than those of the preliminarily heated samples (3 min at 100 degrees C). The heating of the intact fibritin and the mutant G at 50-70 degrees C for 10 min resul ted in the formation of a form with an apparent molecular mass higher than 200 kDa. This form probably represented a trimeric protein with a partly de natured N-terminal part. Fibritins B and C were more stable and were only p artly denatured into monomers even at 70-90 degrees C. The short mutants E and S1 dissociated into monomers at temperatures from 45 to 50 degrees C. T he denaturation of mutants B, C, E, and S1 proceeded in one stage without f ormation of any intermediate form. The stability of the trimeric molecules of native fibritin under PAGE denaturing conditions and the behavior of the intact protein during heating in the temperature range of 50-70 degrees C might be used for the identification of fibritin intermediate forms upon fo lding in vivo. The refolding capability was found for fibritin and its muta nts denatured by heating at low temperatures in the presence of 2% SDS.