JBC, Vol. 250, Issue 17, 6831-6836, Sep, 1975
Chemical and physical studies on the structure of Escherichia coli elongation factor G
M. S. Rohrbach, J. W. Bodley and K. G. Mann
Elongation Factor G (EF-G) from Escherichia coli was purified to homogenity
by a previously published method (Rohrbach, M. S., Dempsey, M. E., and
Bodley, J. W. (1974) J. Biol. Chem. 249, 5094). The protein is composed of
a single polypeptide chain of molecular weight 74,000 under native
conditions and 71,000 under denatured conditions as determined by high
speed equilibrium centrifugation. An apparent molecular weight of 73,000
was found by sodium dodecyl sulfate gel electrophoresis. The protein has an
apparent alpha helix content of 34% as determined from its circular
dichroism spectrum. The extinction coefficient at 280 nm was found to be
62,200 M-1 cm-1. Lysine is the COOH-terminal residue and the sequence at
the NH2 terminus is alanylarginine. No evidence of terminal heterogeneity
was observed. The amino acid composition of EF-G revealed no unusual amino
acids or prosthetic groups, but was notable in that the protein contains
only 6 cysteine residues. The maintenance of at least one of these
cysteines in the reduced form is essential for activity, since the protein
is rapidly inactivated upon removal of protecting thiol. Under some
conditions, activity can be partly restored by re-addition of a thiol. The
per cent activity of the protein was examined by an active site titration,
the formation of the EF-G-ribosome-GDP-fusidic acid complex. The formation
of 1 mol of complex/mol of EF-G showed that the protein is 100% active.
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