The colicin DNase-specific immunity proteins interact with the endonuclease domain of the bacterial toxin colicin E9 with dissociation constants that span the millimolar to femtomolar affinity range. Among the non-cognate interactions Im2 shows the strongest binding toward the E9 DNase domain with a Kd of 10(-8) M, 6 orders of magnitude weaker than that of the cognate immunity protein Im9. Based on a NMR structure of Im9 that shows it to be a 4-helix protein, we have conducted a mutagenic scan in which elements of Im9 secondary structure were substituted into Im2 to precisely delineate regions that define specificity. Eleven chimeras were constructed, and their biological cross-reactivity toward colicins E2 and E9 was evaluated. From this set of mutants seven proteins were purified, and the Kd for their interaction with the E9 DNase domain was measured by a combination of stopped-flow fluorescence and subunit exchange kinetics. Our results show that immunity specificity is dominated by residues on helix II, accounting for 5 orders of magnitude binding specificity relative to Im2, and that packing interactions of helix II with its neighbor helix I and the loop connecting helix III with helix IV play minor roles. The conformational stability of these chimeric proteins was also determined. Proteins displaying an Im9 phenotype were all more stable than the parent Im2 protein, and surprisingly some chimeras were significantly more stable than either Im2 or Im9.