Selective Labeling of Lyslee-Containing Biologically Active Peptides at the N-Terminus with Alexa Fluor Bye

摘要

Pseudomonas aeruginosa readily binds to stainless steel to form highly recalcitrant, organized communities (known as biofilms). These in turn can cause major problems, namely, hospital-acquired infections in susceptible (e.g. immunocompromised or bum) patients with medical implants including catheters, prosthetics and stainless steel implants [1]. P. aeruginosa has polar type IV pili which are made up of thousands of homogenous pilin monomers assembled in a helical array. The pilus-associated epithelial cell receptor binding domain (RBD) is located in the well-structured C-terminal end within residues 128-144 of the C-terminal region of the pilin structural protein of the P. aeruginosa strain PAK as a 14-residue disulfide loop. Binding studies demonstrated that a synthetic peptide of PAK pilin epithelial cell receptor binding domain [PAK(128-144)ox] [(Ac-KCTSDQDEQFIPKGCSK-OH)ox] binds directly to steel with high affinity. The interaction of pili with steel was specifically inhibited by this peptide [1]. Therefore, it seemed reasonable to investigate the binding ability of P. aeruginosa K122-4 wild-type strain RBD peptide [(ACTSNADNKYLPKTCQT-NH2)ox] to stainless steel using fluorescence microscopy. Amine-reactive fluorescent probes are widely used to label peptides at the N-temiinal α-amino group or lysine side-chains. We chose Alexa Fluor 350 to label our K122-4 peptide, because the Alexa Fluor dye conjugates are more readily visualized and more photostable than most other fluorescent conjugates and remain highly fluorescent over a broad pH range [2].