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.
Highlights
Environment-sensitive fluorophores are fluorophores, the excitation spectra, emission spectra and/or quantum yields (QY) of which depend on variables such as pH, solvent polarity, viscosity and even molecular crowding/aggregation state
Solvatochromic fluorophores, the λmax of which changes with solvent polarity, are often push-pull molecules with an electron-donating and an electronwithdrawing group bound to an aromatic moiety
cationic antimicrobial peptides (CAMP) often show additional pharmacologically relevant biological activities, like antifungal, antitumor and anti-inflammatory activity [23,24]. p53pAnt is a designed antitumor peptide containing a sequence at the N-terminus that induces apoptosis by interacting with p53, as well as a “cell-penetrating peptide” (CPP) at the C-terminus that derived from the Antennapedia protein homeodomain, which can drive peptides inside eukaryotic cells both by directly crossing the cytoplasmic membrane and by mediating endocytosis [25,26,27]
Summary
Environment-sensitive fluorophores are fluorophores, the excitation spectra, emission spectra and/or quantum yields (QY) of which depend on variables such as pH, solvent polarity, viscosity and even molecular crowding/aggregation state. Several different molecular mechanisms can contribute to modulation of the fluorescence of environmentsensitive fluorophores [1]. Solvatochromic fluorophores, the λmax of which changes with solvent polarity, are often push-pull molecules with an electron-donating and an electronwithdrawing group bound to an aromatic moiety. Solvent polarity strongly affects the intramolecular charge transfer of these molecules, modulating their fluorescence [1]. In a less common type of solvatochromic fluorophores, an excited-state intramolecular proton-transfer event generates tautomeric forms with different absorption and emission spectra [1]. Conformational changes or variations in the aggregation state strongly influence the fluorescence of the probe [1].
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