LL‐37 is an innate immune peptide derived from the human cathelicidin, which exerts pleiotropic roles in host defense and healing. These activities in part depend on its capacity to adopt an amphipathic helical structure in physiological solutions and then oligomerizing. Orthologues from other primates, such as rhesus RL‐37, remain monomeric and disordered under the same conditions. Intramolecular salt‐bridges, arising from appropriately spaced anionic and cationic residues in its sequence, may play a relevant role in determining the particular structure adopted by LL‐37. To probe this, we have effected minimal, targeted residue variations such as replacement of a single residue (K15→G), or inversion of one or both sets of two residues (E10 K11→ K10 E11 or E16 K18→ K16 E18). This could alter the pattern of intramolecular salt bridging without affecting other functionally relevant parameters such as overall hydrophobicity, helix amphipathicity or charge. The structural and functional effects were analyzed using CD spectroscopy, surface plasmon resonance, antimicrobial activity assays, and bacterial membrane permeabilization to fluorescent probes of increasing sizes, using flow cytometry. Analogs were functionally different from both LL‐37 and RL‐37, so it was not possible to switch from the function of one to that of the other simply by altering the salt‐bridging pattern in this manner. This indicates that the particular structure/function characteristics of LL‐37 likely depend quite subtly, and in a precise and complex manner, on a complex pattern of intramolecular interactions.
