EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE G-DOMAIN OF SACCHAROMYCES-CEREVISIAE ELONGATION-FACTOR-2

Protein synthesis elongation factor 2 (EF-2), a member of the G protei n superfamily, catalyzes the movement of the ribosome along mRNA in a reaction driven by the hydrolysis of GTP. In the present study, we hav e expressed, purified, and characterized the G domain of Saccharomyces cerevisiae EF-2 to define its functional properties. A peptide of 185 amino acids was found to be sufficient to bind guanine nucleotides wh en expressed in Escherichia coli either as a fusion with the maltose b inding protein or as a truncation. The fusion was expressed as a solub le, active protein, while the truncation was expressed as an insoluble protein that required renaturation in order to function, Similar to n ative EF-2, both expression products bound GDP, but they did so with s ignificantly lower affinity (K-d ca. 200 mu M for both the fusion and the truncation vs ca. 0.5 mu M for native EF-2). However, in contrast to native EF-2 which requires the ribosome for GTP hydrolysis, the iso lated G domain hydrolyzed GTP (K-m ca. 40 mu M; turnover rate ca. 0.5 min(-1)) in the absence of the ribosome. We conclude that the G domain residues necessary for GTP binding and hydrolysis are located in the first 185 amino acids of S. cerevisiae EF-2. (C) 1996 Academic Press, Inc.