(A) Structure of the antimicrobial peptide cWFW with colour-highlighted aromatic residues that were individually substituted by the fluorescent label in this study; (B) the fluorescent amino acid FHC used as the label (framed and colored) along with potential alternatives, i.e., reported fluorescent amino acids with the excitation wavelengths higher than 360 nm. Compounds are designated according to original reports, excitation wavelengths (?Ex.?) are stated; (C) excited-state intramolecular proton transfer (ESIPT)-mediated fluorescence, causing dual emission and environmental sensitivity of the chromophore in 3-hydroxychromone-containing labels. N (N*) and T (T*) represent normal and tautomeric forms, respectively, in the ground (excited) state; BPT denotes back proton transfer in the ground state.

CD spectra of the peptides in various environments (top to bottom traces): in 10 mM phosphate buffer, pH 7,4, in 2,2,2-trifluoroethanol (TFE), in 10 mM sodium dodecyl sulphate (SDS) micelles at a peptide/detergent ratio (P/D) of 1/100, in the presence of large unilamellar vesicles (LUVs) modeling an “animal” (DMPC/cholesterol, 4/1) or a “bacterial” (DMPC/DMPG, 1/1) plasma membrane in terms of the lipid composition. The spectra were acquired from fresh 100 µM (or 200 µM for LUVs) peptide solutions at 25°C or 35°C (LUVs). P/L designates peptide/lipid ratio.

(A) Planktonic bacterial growth inhibition according to a broth microdilution assay. Numbers depict µM values calculated from µg/ml dilutions; (B) concentration-dependent hemoglobin release from human erythrocyte suspension; and (C) fluorescence microscopy observation of Hoechst 33342 (blue color)-co-stained Hela cells after 1 h of incubation with 30 µM FHC-labelled cWFW analogues (green color). Images are 105 × 114 µm.

Fluorescence spectra (not normalized) from 8 µM solutions of the FHC-labeled cWFW analogues in H₂O and DMF under different excitation wavelengths (indicated as “Ex 370 nm” and “Ex 405 nm”).

Intensity-normalized absorbance spectra from 8 µM solutions of the FHC-labelled cWFW analogues in solvents of different polarities, colored according to the polarity index (Reichardt and Welton, 2019, insert, “a)”). Black arrows mark excitation wavelengths used for fluorescence measurements and microscopy (370 and 405 nm).

Orthogonal projections of the two energetically preferred conformations in vacuo of the peptide cRW-F²/FHC, according to an MM+ molecular modeling run: (A) “closed” state, where all three arginyls interact with the FHC side-chain, and (B) “open” state, where the arginyl cluster and the aromatic side-chains are well separated. The FHC residue is in green and CPK. Trp¹ and Trp³ are in dark and light blue, respectively. The residues interacting with FHC are shown in CPK rendering mode.

Band-filtered confocal fluorescence images of adherent HeLa and HEK293 cell cultures incubated with 30 µM cRW-W³/FHC for 5 and 60 min (indicated). Spectral ranges for collecting emission data are shown at the bottom of the image stacks; BF = bright-field images.

Cytotoxicity evaluation of cWFW and its fluorescent analogues by fluorescence microscopy. (A) First, at higher magnification (63× objective), the cells were imaged to monitor cell entry of the peptides; HeLa cells with 20 µM peptide imaging after 1 h of incubation are shown for illustration. (B) After imaging (2.5 h after peptide addition), Hoechst/PI co-staining is applied and imaged at 20× magnification, exemplified here by experiments with HEK293 cells; (C) Total/dead cell counting results for all peptides at 20 µM concentration co-incubated with both cell types.

Orthogonal section views from four dpf zebrafish embryos injected with either cWFW in PBS (A–D) or cRW-W³/FHC peptide (E–H). The trunk region was imaged by detecting emitted fluorescence light in a range of either 415–435 nm (A, C, E, G) or 470–520 nm (B, D, F, H) after sequential two-photon excitation at 730 nm (A, B, E, F) and 810 nm (C, D, G, H). Sagittal sections (A–H), transverse sections (A′–H′) and horizontal sections (A′′–H′′) are shown. Dashed lines (magenta) in sagittal views indicate the position where horizontal views were made. Arrows indicate the caudal artery, where cRW-W³/FHC and other peptides were mainly found to be localized. Scale bar: 50 µm.