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Protein Science, Vol 7, Issue 12 2595-2601, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

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

U. S. LADROR, D. A. EGAN, S. W. SNYDER, J. O. CAPOBIANCO, R. C. GOLDMAN, S. A. DORWIN, R. W. JOHNSON, R. EDALJI, A. V. SARTHY, T. McGONIGAL, K. A. WALTER and T. F. HOLZMAN
Pharmaceutical Discovery Research, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39{deg}C, and a minor transition at 58{deg}C. Ultracentrifugation shows that EF-3 is a monomer; thus, 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, generating 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 ~75 kDa and the other at ~55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58{deg}C transition, and a loose structure of the C-terminus, giving rise to the 39{deg}C transition. Three potentially functional domains of the protein 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 trypsin, but the ribosome-stimulated activity was inactivated faster.
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R. Kambampati, C. Pellegrino, A. Paiva, L. Huang, L. Mende-Mueller, and K. Chakraburtty
Limited Proteolysis of Yeast Elongation Factor 3. SEQUENCE AND LOCATION OF THE SUBDOMAINS
J. Biol. Chem., May 26, 2000; 275(22): 16963 - 16968.
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