Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues

Abstract

Environment-sensitive fluorophores are very valuable tools in the study of molecular and cellular processes. When used to label proteins and peptides, they allow for the monitoring of even small variations in the local microenvironment, thus acting as reporters of conformational variations and binding events. Luciferin and aminoluciferin, well known substrates of firefly luciferase, are environment-sensitive fluorophores with unusual and still-unexploited properties. Both fluorophores show strong solvatochromism. Moreover, luciferin fluorescence is influenced by pH and water abundance. These features allow to detect local variations of pH, solvent polarity and local water concentration, even when they occur simultaneously, by analyzing excitation and emission spectra. Here, we describe the characterization of (amino)luciferin-labeled derivatives of four bioactive peptides: the antimicrobial peptides GKY20 and ApoBL, the antitumor peptide p53pAnt and the integrin-binding peptide RGD. The two probes allowed for the study of the interaction of the peptides with model membranes, SDS micelles, lipopolysaccharide micelles and Escherichia coli cells. Kd values and binding stoichiometries for lipopolysaccharide were also determined. Aminoluciferin also proved to be very well-suited to confocal laser scanning microscopy. Overall, the characterization of the labeled peptides demonstrates that luciferin and aminoluciferin are previously neglected environment-sensitive labels with widespread potential applications in the study of proteins and peptides.

The Antimicrobial, Antibiofilm and Anti-Inflammatory Activities of P13#1, a Cathelicidin-like Achiral Peptoid

“…In the presence of SDS micelles, in addition to the major peak in the green region due to the phenolate, the emission spectra also show a broad peak in the blue region (430-450 nm). This emission, which is typical of the neutral form of luciferin (Figure S3 in Supplementary Materials), can only be observed in environments with very low water contents and no alternative proton acceptors, the sole conditions that can hinder the photoinduced deprotonation process [70], and confirms that the fluorophore is buried into the micelles. Furthermore, the blue shift of the phenolate emission (5-10 nm) confirms that the fluorophore is in a less polar environment.…”

“…We recently developed a peptide-labelling strategy based on an environment-dependent luciferin-like fluorophore [70]. The amino and hydroxy versions of this fluorophore are strongly sensitive to environmental polarity and proton acceptor availability and/or local pH, respectively.…”

“…Hence, we studied the interactions of the labeled P13#1 variants with so-dium dodecyl sulfate (SDS) micelles, LPSs (free and micellar) and whole bacterial cells. The previously characterized labeled variants of GKY20, Luc-and aLuc-GKY20 [70], were used as controls. The excitation and emission spectra of Luc-and aLuc-P13#1 in the presence of SDS, LPSs and bacterial cells (E. coli ATCC 25922, P. aeruginosa PAO1 and S. aureus ATCC 6538P) were very similar to those of Luc-and aLuc-GKY20, respectively, thus demonstrating that the binding properties of GKY20 and P13#1 are comparable.…”

“…The fitting procedure The binding of the P13#1 variants to SDS, the LPSs and the bacterial cells was also accompanied by strong positive or negative variations in the emission intensity. Usually, solvatochromic fluorophores show higher emission intensities in apolar environments, but luciferins are also sensitive to quenching, e.g., intramolecular, conformation-dependent quenching from tryptophan and tyrosine [70]. Therefore, the observed variations in the fluorescence emission are a net result deriving from these two opposite contributions.…”

“…CG-P13#1 (0.5 mg/mL final concentration) was labeled using 6-hydroxy-2-cyanobenzo thiazole (CBT-OH) or 6-amino-2-cyanobenzothiazole (CBT-NH2) to obtain, respectively, Luc-and aLuc-P13#1. The labeling was performed following the procedure previously described in [70,79], with minor modifications. Briefly, reactions with cyanobenzothiazoles (CBTs) were performed in a 15 mM sodium phosphate buffer (NaP) at a pH of 7.4 which contained 2 M guanidine-HCl, 1 mM Tris(2-carboxyethyl)phosphine (TCEP) and 1 mM CBTs (10× stock solutions in DMF).…”