Domain structure analysis of elongation factor-3 fromsaccharomyces cerevisiaeby limited proteolysis and differential scanning calorimetry
Autor: | Robert C. Goldman, Seth W. Snyder, Tom Mcgonigal, Karl A. Walter, Uri S. Ladror, John O. Capobianco, Sarah A. Dorwin, Rohinton Edalji, David A. Egan, Thomas F. Holzman, Robert W. Johnson, Aparna V. Sarthy |
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Rok vydání: | 1998 |
Předmět: |
Saccharomyces cerevisiae Proteins
Proteolysis Molecular Sequence Data Saccharomyces cerevisiae Biology Biochemistry Fungal Proteins Differential scanning calorimetry medicine Trypsin Amino Acid Sequence Molecular Biology Peptide sequence Chromatography High Pressure Liquid Fungal protein Calorimetry Differential Scanning medicine.diagnostic_test Peptide Elongation Factors biology.organism_classification Peptide Fragments Elongation factor Electrophoresis Polyacrylamide Gel Ultracentrifuge Ultracentrifugation Research Article medicine.drug |
Zdroj: | Protein Science. 7:2595-2601 |
ISSN: | 1469-896X 0961-8368 |
Popis: | 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 degrees C, and a minor transition at 58 degrees 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 approximately 75 kDa and the other at approximately 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 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees 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. |
Databáze: | OpenAIRE |
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