New aspects of the structure and mode of action of the human cathelicidin LL-37 revealed by the intrinsic probe p-cyanophenylalanine.
The human cathelicidin peptide LL-37 is an important effector of our innate immune system and contributes to host defence with direct antimicrobial activity and immunomodulatory properties, and by stimulating wound healing. Its sequence has evolved to confer specific structural characteristics that strongly affect these biological activities, and differentiate it from orthologues of other primate species. In the present paper we report a detailed study of the folding and self-assembly of this peptide in comparison with rhesus monkey peptide RL-37, taking into account the different stages of its trajectory from bulk solution to contact with, and insertion into, biological membranes. Phenylalanine residues in different positions throughout the native sequences of LL-37 and RL-37 were systematically replaced with the non-invasive fluorescent and IR probe p-cyanophenylalanine. Steady-state and time-resolved fluorescence studies showed that LL-37, in contrast to RL-37, forms oligomers with a loose hydrophobic core in physiological solutions, which persist in the presence of biological membranes. Fourier transform IR and surface plasmon resonance studies also indicated different modes of interaction for LL-37 and RL-37 with anionic and neutral membranes. This correlated with a distinctly different mode of bacterial membrane permeabilization, as determined using a flow cytometric method involving impermeant fluorescent dyes linked to polymers of defined sizes.
*Department of Life Sciences, University of Trieste, Trieste, Italy.
Daniela
D
Xhindoli
*Department of Life Sciences, University of Trieste, Trieste, Italy.
Francesca
F
Morgera
¶Department of Chemistry, Technical University of Munich, Garching, Germany.
Ursula
U
Zinth
*Department of Life Sciences, University of Trieste, Trieste, Italy.
Roberto
R
Rizzo
*Department of Life Sciences, University of Trieste, Trieste, Italy.
Sabrina
S
Pacor
*Department of Life Sciences, University of Trieste, Trieste, Italy.
Alessandro
A
Tossi
D000409:Alanine / Q000031:analogs & derivatives* / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D002645:Chickens; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D008251:Macaca; D008969:Molecular Sequence Data; D009570:Nitriles / Q000032:analysis*; D011487:Protein Conformation; D054804:Cathelicidins; D010649:Phenylalanine / Q000031:analogs & derivatives
D000409:Alanine; D023181:Antimicrobial Cationic Peptides; D005456:Fluorescent Dyes; D009570:Nitriles; D054804:Cathelicidins; C530634:p-cyanophenylalanine; D010649:Phenylalanine