On the global architecture of initiation factor IF3: a comparative study of the linker regions from the Escherichia coli protein and the Bacillus stearothermophilus protein.

Initiation factor IF3 is a protein involved in the initiation stage of protein synthesis. It consists of two global domains linked by a 20 residue long, solvent-exposed linker. Recently, the structure of the N and C-terminal domains of the Bacillus stearothermophilus protein have been solved by X-ray crystallography and the structure of the intact Escherichia coli protein has been studied by NMR. These two studies have led to apparently contradictory models for the domain organization of IF3. The NMR study of the E. coli protein indicates that the linker region is flexible, while the studies of the isolated N and C-terminal domains of the B. stearothermophilus protein suggest that the linker forms a rigid helical rod. In order to resolve this discrepancy, a set of peptides corresponding to the linker regions of the B. stearothermophilus and the E. coli protein were synthesized. Circular dichroism and NMR spectroscopy were used to study the helical content as a function of pH, temperature, peptide concentration and ionic strength. Both peptides are monomeric. The estimated helical content of the linker fragment from B. stearothermophilus is 68% at high pH and 1 degree C. The measured helicity decreases to 53% at pH 7.0 and 1 degree C. In contrast, the peptide corresponding to the E. coli IF3 linker region is largely unstructured with a maximum helical content of 15% at high pH and only 8% at pH 7.0, 1 degree C. These results suggest that the different structures observed for the two intact proteins may be due to the different intrinsic stability of the two linker peptides. The helical content of the two linker peptides is, however, much closer when the peptides are compared at the respective temperatures of optimum growth for E. coli and B. stearothermophilus (3% versus 17%). The pH and ionic strength dependence of the helical content of the B. stearothermophilus peptide demonstrates that side-chain/side-chain interactions play an important role in stabilizing the helical structure. In addition, studies with mutant peptides show that the first Asp residue in the linker sequence helps to stabilize the helix via an N- capping interaction.