Structure-function relationship in Escherichia coli initiation factors : Environment of the Cys residue and evidence for a hydrophobic region in initiation factor IF3 by fluorescence and ESR spectroscopy

Abstract

Escherichia coli translational initiation factor IF3 contains a single Cys residue at position 66 of its primary structure. The environment and the relevance of this residue for the biological activity of the factor has been investigated by the use of several -SH group reagents, some of them carrying ESR or fluorescent probes. It was found that: (a) The Cys residue in itself is not essential for activity since its modification with N-ethylmaleimide, iodoacetamide, fluorescein maleimide, and the spin-label tetramethylpiperidinoxyl maleimide resulted in little or no loss of biological activity. (b) The Cys residue is exposed and reactive in native unbound protein as well as in 30-S-bound IF3, although the rate, but not the extent, of reaction is somewhat affected by the state of the protein (faster in urea-denatured IF3 and slower in 30-S-bound IF3). (c) ESR spectroscopy of spin-labeled IF3 showed that the SH-bound probe is weakly immobilized and located in a hydrophilic environment in the native-free protein; upon binding to 30-S ribosomal subunits, however, the probe becomes more immobilized. High concentrations of salt reverse this effect, which is not observed upon binding of IF3 to the synthetic polynucleotide poly(AUG) or trinucleotide (AUG). (d) Formation of an adduct between the Cys residue and four different maleimide derivatives bearing hydrophobic fluorescent probes (e.g., anilinonaphthyl maleimide) results in the inactivation of the factor. Molecular dimensions and spectral properties suggest that the fluorescent probe reaches and becomes immobilized in a hydrophobic region (patch or pocket) situated within approximately 8 Å of the SH group; energy transfer from Tyr residues also suggests that the hydrophobic region contains or is close to at least one Tyr residue, (e) The same hydrophobic region probably constitutes the primary binding region where the hydrophobic probe anilinonaphthylsulfonate (the chromophore of anilinonaphthyl maleimide) binds noncovalently to the IF3 molecule, producing an increase in fluorescence quantum yield and an inactivation similar to that caused by anilinonaphthyl maleimide. Taken together, we interpret these results to indicate that although the Cys residue is found in a hydrophilic environment and is not essential for ribosomal binding of the factor, it is located near the edge of the binding site, within a short distance of a hydrophobic region essential for activity.