Difference FTIR studies reveal nitrogen-containing amino acid side chains are involved in the allosteric regulation of RecA.

The Escherichia coli RecA protein performs the DNA strand-exchange reaction utilized in both genetic recombination and DNA repair. The binding of nucleotides triggers conformational changes throughout the protein resulting in the RecA-ATP (high DNA affinity) and RecA-ADP (low DNA affinity) structures. Difference infrared spectroscopy has allowed us to study protein structural changes in RecA that occur after binding ADP or ATP. Experiments were performed on control and uniformly (15)N-labeled RecA in an effort to assign vibrational changes to protein structures and study the molecular changes associated with the allosteric regulation of RecA. Comparison of RecA-ATP and RecA-ADP data indicates that the protein adopts unique secondary structures in each form and altered N-H stretching vibrations in the RecA-ADP structure not observed in the RecA-ATP data. Numerous vibrations throughout the 1700-1300 cm(-)(1) region are influenced by isotopic substitution and imply that many nitrogen-containing side chains are altered after ADP binds to RecA. The RecA-ATP data contain unique vibrations that are not observed in the RecA-ADP data and may be associated with Gln, Lys, Arg, or Asn. Model compound studies on control and (15)N-labeled glutamine and lysine provide additional evidence that supports the tentative assignments of vibrations observed in our difference spectra. In addition, we provide evidence that nitrogen-containing amino acids are important in locking in the low-DNA affinity, more compact conformation of the protein and that some of these interactions may not be present in a more extended, flexible RecA-ATP conformation.