Domain structure analysis of elongation factor-3 from Saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein syn thesis machinery. In this communication the structure of EF-3 from Saccharo myces cerevisiae is characterized by differential scanning calorimetry (DSC ), ultracentrifugation, and limited tryptic digestion. DSC shows a major tr ansition at a relatively low temperature of 39 degrees C, and a minor trans ition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; th us, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, gene rating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N -terminal fragment slowly undergoes further digestion generating two major bands, one at similar to 75 kDa and the other at similar to 55 kDa. The lat ter was unusually resistant to further tryptic digestion. The 33-kDa C-term inal fragment was highly sensitive to tryptic digestion. A 30-min tryptic d igest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results su ggest a tight structure of the N-terminus, which may give rise to the 58 de grees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the p rotein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622 ), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by try psin, but the ribosome-stimulated activity was inactivated faster.