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 protein superfamily, catalyzes the movement of the ribosome along mRNA in a reaction driven by the hydrolysis of GTP. In the present study, we have 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 when expressed in Escherichia coli either as a fusion with the maltose binding protein or as a truncation. The fusion was expressed as a soluble, active protein, while the truncation was expressed as an insoluble protein that required renaturation in order to function. Similar to native EF-2, both expression products bound GDP, but they did so with significantly lower affinity (Kd ca. 200 microM for both the fusion and the truncation vs ca. 0.5 microM for native EF-2). However, in contrast to native EF-2 which requires the ribosome for GTP hydrolysis, the isolated G domain hydrolyzed GTP (Km ca. 40 microM; 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.
Rao
S
S
Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota, 55455, USA.
Bodley
J W
JW
D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000235:genetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D017931:DNA Primers; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D007544:Isopropyl Thiogalactoside; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins