PQS (2-heptyl-3-hydroxy-4(1H)-quinolone) was purchased from Sigma-Aldrich (product no. 94398, purity 99.8%). C-terminal amidated LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES-NH₂, cat. no. 308114) was synthesized by NovoPro Bioscience Inc. (Shanghai, China). Peptide purity corresponded to 96.20%. The molecular weight of the peptide (4492.25) was determined by mass spectrometry. The net peptide content of the supplied material was 72.12%. All other chemicals were of analytical reagent grade.

LL-37 solutions were prepared in pH 7.4 phosphate-buffered saline (PBS) or in 10 mM Tris-HCl buffer (pH 7.4). PBS was prepared by dissolving 8 g of NaCl (137 mM), 0.2 g of KCl (2.7 mM), 1.8 g of Na₂HPO₄·2H₂O (10 mM), and 0.312 g of NaH₂PO₄·2H₂O (2 mM) in 1 L of Milli-Q water. PQS was dissolved in spectroscopic grade ethanol.

Circular dichroism (CD) and UV/VIS absorption spectra were recorded on a JASCO J-715 spectropolarimeter at 25 ± 0.2°C in a 0.2 cm path-length rectangular quartz cuvette (Hellma, United States). Temperature control was provided using a Peltier thermostat. CD data as ellipticities in mdeg were collected in a continuous scanning mode between 200 and 460 nm at a rate of 100 nm/min, with a step size of 0.1 nm, a response time of 2 s, three accumulations, and a 1 nm bandwidth. CD curves of peptide and PQS-peptide samples were corrected by the spectral contribution of the blank PBS and the free LL-37, respectively. For CD titration measurements, 400 μL of LL-37 solution (33–34 μM) was placed in the cuvette. After finishing the CD scans, μL aliquots of the PQS stock solution were pipetted consecutively into the cuvette and the CD data were collected after each aliquot. For the evaluation of the structural effect of ethanol added with PQS during the titration procedure, the far-UV CD curves of LL-37 were measured at increasing ethanol concentrations. UV/VIS absorption spectra of PQS in the absence and presence of LL-37 were recorded by conversion of the high-tension (HT) values of the photomultiplier tube of the CD equipment into absorbance units. All CD and absorption spectroscopic measurements were implemented once.

UV/VIS spectra were recorded using a Hewlett-Packard 8453 diode array spectrophotometer at 25°C in PBS between 190 and 600 nm in 1 nm increments using a quartz cuvette of a 1 cm path length. The titration experiment was performed by a consecutive increase of PQS concentration up to 15 μM. All spectra were baseline-corrected with the spectrum of PBS. UV/VIS spectroscopic measurements were implemented twice.

The average size, size distribution, and time-dependent autocorrelation functions of the formed aggregates were evaluated using a W130i apparatus (Avid Nano Ltd., High Wycombe, United Kingdom). Samples containing a constant peptide concentration (34 μM) and various amounts of added PQS were measured in PBS in a final volume of 200 μL in low-volume disposable cuvettes (UVette, Eppendorf Austria GmbH). The analyses of the measurements were performed using the i-Size 3.0 software supplied with the device.

All computations were carried out using the Gaussian 09 software package (Frisch et al., 2004). To optimize CPU requirements, the PQS molecule was truncated, keeping only its aromatic hydroxyquinolone moiety. The structure was optimized at the ωB97X-D/6-311++G (2d, 2p) level of theory. To aid identification of which electronic transition dipole moment (etdm) belongs to which band in the experimentally observed spectra as well as to obtain the orientation of these moments within the molecule, excited energies and UV/VIS spectra of the optimized geometry were calculated using time-dependent density functional theory (TD-DFT) calculations at the same level of theory as indicated above. Accordingly, the three etdms with the highest oscillator strength were found at 341.04, 239.68, and 223.05 nm.

The planar, hydroxyquinolone chromophore of PQS is responsible for the multicomponent absorption band system measured in PBS above 200 nm (Figure 1A). The most intense peak is centered at 245 nm overlapping with two adjacent, weaker bands at both the shorter and longer-wavelength sides. A broad, less intense band is located between 300 and 370 nm decorated with two, partially resolved maxima. In relation to the buffer solution, a similar absorption spectroscopic profile was obtained in ethanol with some differences to be noted. In the organic solvent, a moderate red shift of the λ _max values can be observed. Additionally, no absorption band is displayed either above 375 nm or between 260 and 280 nm (Figure 1A). Taking into consideration the structure of PQS as well as its molar absorption intensities (ε _max), the UV absorption bands can be assigned to the π–π ^* transitions of the conjugated π-system of the planar hydroxyquinolone moiety. To determine the respective electronic transition dipole moments (etdms) associated with the main absorption peaks, TD-DFT quantum chemical calculations were performed. According to the results, the etdm of the higher oscillator strength 240 nm band lies along approximately the long axis of the ring system, whereas for the longer-wavelength transition, it is oriented at 70° relative to the direction of the 240 nm etdm (Figure 2).

Upon increasing the PQS concentration above 3 μM, the UV/VIS spectra exhibited some notable alterations. At the red edge of the spectrum, the zero baseline started to elevate, and it is obvious that the absorbance values do not follow the Beer–Lambert law (Figure 1B). The amplitude of the peak at 245 nm hardly increases with the concentration, whereas the unresolved band around 260 nm gained double intensity. Similarly, the longest-wavelength weak and broad band above 360 nm strongly increased and finally became equal in magnitude with the adjacent peak. In concert with these changes, increased opalescence of the sample solution could be noticed. In view of the observations, the anomalous absorption spectroscopic features can be attributed to the concentration-dependent, progressive aqueous aggregation of the hydrophobic PQS molecules. Associative interactions between planar organic compounds possessing limited water solubility such as acridine (Ikkda and Imae 1971) and cyanine dyes (Kim et al., 2006; Wurthner et al., 2011), carotenoids (Zsila et al., 2001), and porphyrin derivatives (Kubat et al., 2003) occur frequently in aqueous solutions. Depending on the particular chemical constitution of the molecules, the resulting dimers, oligomers, and higher-order aggregates may be stabilized by π–π stacking and attractive coulombic and/or van deer Waals forces. Packing of the aromatic rings within PQS associates results in the delocalization of the π–π ^* excited states over a wide range of the adjacent chromophores. Thus, electronic absorption spectra of such optically excited aggregate systems display characteristic signs due to the intermolecular electronic interaction between the excited states termed as exciton coupling (Kasha 1963; Kasha et al., 1965; Boiadjiev and Lightner 2005). In relation to the absorption features of the monomeric state, spectral changes caused by exciton coupling include hypo- or hyperchromism of existing bands, red or blue shift of the λ _max values, band broadening/narrowing, and/or the development of one or more additional peaks. Depending on the relative arrangement of the molecules established inside the assemblies, the resulting spectral signatures may indicate the existence of J- or H-type aggregation (Zsila et al., 2001; Kim et al., 2006; Wurthner et al., 2011; Pescitelli et al., 2014). Compared to the monomeric compound, J-aggregates exhibit a new, red-shifted, narrow, and intense band due to the parallely displaced, head-to-tail-type geometry of the chromophores enabling intermolecular excitonic delocalization between them (Scheme 2). In concert with this, the evolution of new, red-shifted peaks of PQS centered at 265 and 377 nm (Figure 1B) is indicative of the presence of J-aggregating motifs composed of hydroxyquinolone moieties. A hypothetical J-dimer of two PQS molecules is shown in Scheme 2 stabilized by CH–π interactions formed between the alkyl chain and the π-system of the hydroxyquinolone rings. Note that this kind of arrangement ensures exciton coupling between the head-to-tail oriented etdms, resulting in red-shifted J-bands in the absorption spectrum at 265 and 377 nm (Figure 1B). The steadily growing light scattering of the solution as well as the spectral overlap with the still significant monomeric absorption render these new bands in part unresolved. The weak bands in the UV/VIS spectrum at 264 and 377 nm recorded even at the lowest PQS concentration suggest that a minor fraction of the molecules are already in an aggregated state even in dilute solution (Figure 1A). The evaluation of the absorption curve measured in ethanol lends further credence to this assumption. The complete absence of these bands indicates pure monomeric state of PQS in that solvent.

Further on, UV/VIS spectroscopic measurements were performed to determine the critical aggregation concentration of PQS in PBS solution. At low concentrations (∼1–2 μM) only monomers are present in the buffer solution (Supplementary Figure S1). With increasing concentration, the J-band appears with a maximum at 376 nm which could be assigned to the aggregate formation. Considering the UV signal enhancement, titration data showed nearly linear dose dependence, with a breakpoint in the lower-concentration range (Supplementary Figure S1, inset). Similar to micellar systems, PQS molecules dissolved in PBS are monomeric below the critical aggregation concentration (cac), an important parameter that characterizes the self-assembling behavior of such systems. The cac value was estimated from the discontinuity in the absorbance intensities at 376 nm, resulting in a 2.94 ± 0.34 μM value in PBS (Supplementary Figure S1, inset).

Evaluation of the far-UV CD spectra is an excellent and rapid tool to obtain valuable information about conformational features of the peptide backbone, that is, the relative contribution of the most important secondary structural elements such as α-helix, β-sheet, and random coils (Toniolo et al., 2012). Therefore, the secondary solution structure of LL-37 was investigated by CD spectroscopy. In line with previous reports (Brannon et al., 2015; Johansson et al., 1998) and consistent with the two negative extrema at 209 and 223 nm, the far-UV CD spectrum of LL-37 obtained in PBS solution refers to the ordered, primarily α-helical conformation of the peptide chains (Figure 3). In contrast, the CD curve recorded in a salt-free Tris-HCl buffer (pH 7.4) lacks the helix-like, similar intensity double minima and shows a main negative peak of π–π ^* origin centered at 203 nm, whereas the n–π ^* transitions of the amide chromophores produce a weaker, broad band having a minimum around 225 nm (Figure 3). This CD pattern is fairly typical of a predominantly disordered state with a minimal helix content (Johansson et al., 1998; Zsila et al., 2019). Accordingly, the α-helical structure of LL-37 maintained in PBS is completely disrupted in the absence of inorganic anions. The balance of intrapeptide ionic attractions–repulsions between charged residues (arginine/lysine and glutamate/aspartate) might be a decisive factor in determination of the helix-coil equilibrium, particularly for sequences rich in acidic and basic amino acids. Salt bridges formed by acidic and basic residues located in positions i and i + 3 or i + 4, which are close on the α-helix, specifically stabilize the helical conformation (Johansson et al., 1998; Scholtz et al., 1993). Vice versa, side chains with the same charge located at these sites destabilize the helix due to the repulsive electrostatic interactions. The sequence of LL-37 contains nine potential helix-promoting oppositely charged ion pairs and seven combinations of basic residues located both in positions i and i + 3 or i + 4 (Figure 3). As inferred from CD spectroscopic data, the presence of various anions in a suitable concentration shifts the conformational equilibrium of LL-37 toward the ordered, helical form (Johansson et al., 1998; Zsila et al., 2019). The anion-induced folding may occur via coulombic shielding of the cationic side chains, thereby reducing the repulsion forces between them.

Since PQS is achiral, neither its monomeric form (in EtOH) nor its self-associated form (in buffer) exhibits CD signals (data not shown). Surprisingly, upon addition of PQS to LL-37 dissolved in PBS, well-resolved multiple CD bands were induced (Figure 4). The respective absorption bands to which these signals are allied bear the typical spectroscopic features of the aggregated state. Similar to that observed in a peptide-free buffer solution, two new peaks appeared around 260 and 375 nm and the long-wavelength tail of the absorption curve rose strongly with the increase of PQS concentration. The band developed at 375 nm splits into two intense Cotton effects (CEs) of opposite signs in the CD spectrum, changing from negative to positive close to the absorption maximum (Figure 4). At shorter wavelengths, a similar but oppositely signed and less intense CD couplet is shown associated with the UV maximum at 260 nm. Additional negative signals are displayed around 248 and 230 nm. The magnitude of the induced CEs increases with the PQS concentration of the sample solution but cannot be saturated even at 15-fold molar excess of the ligand molecules. According to the induced CD pattern observed above 240 nm, the peptide chains serve as asymmetric templates for PQS molecules to build up chiral supramolecular aggregates. It is reasonable to assume that in the initial phase, the association of J-aggregated PQS dimers/oligomers to LL-37 occurs in a chiral fashion dictated by the asymmetric centers of the side chains. In parallel with the advance of ligand loading, the size of the chiral-arranged associates progressively increases by the adsorption of more and more apolar signal molecules. It should be noted that the parallel alignment of adjacent PQS molecules (Scheme 2) lacks any chiral organization. Thus, it is proposed that the induced CD activity stems from the twist between J-aggregated building blocks deposited onto the chiral surface of LL-37. According to this mechanism, chiral exciton coupling between such helically disposed J-aggregated dimers or oligomers is responsible for the bisignate CEs observed experimentally (Kim et al., 2006). The helical sense of the peptide-templated self-assemblies can be deduced from the exciton chirality rule (Boiadjiev and Lightner 2005; Pescitelli et al., 2014). The in-phase and out-of-phase exciton coupling of the individual π–π ^* transition moments of hydroxyquinolone chromophores arranged helically relative to each other in the assembly generate two bands, a higher- and a lower-energy oppositely signed CD band. The sign order of these exciton CEs is governed by the relative orientation of the interacting transition moments: a clockwise screw sense between them leads to a longer-wavelength positive CD band and a shorter-wavelength negative CD band and vice versa. Taking this into consideration, the main bisignate CD feature allied to the J-band in the near-UV region suggests the prevalence of counterclockwise twist between the interacting transition dipole moments oriented along the long axis of the hydroxyquinolone ring. In view of the shorter-wavelength opposite CD couplet, the large deviation of its respective etdm from that of the 340 nm band must be considered (Figure 2). This implies that various intermolecular arrangements exist where these etdms are coupled just in an opposite sense in relation to the longer-wavelength dipole moments (Scheme 3). Consequently, the prevalence of these species can be accounted for the reversed, negative–positive and positive–negative exciton couplets measured for the J-absorption band at 375 and 261 nm, respectively (Figure 4).

Besides the exciton signals, an additional positive CE is displayed at 354 nm. It coincides with the UV band centered at the same wavelength which represents the light absorption of the monomeric PQS species (Figure 4). Accordingly, a monomeric fraction of PQS is also involved in the peptide binding and thus gives contribution to the induced CD activity presumably via the non-degenerate exciton coupling mechanism (Zsila et al., 2011).

It is tempting to speculate that the supramolecular chirality of the J-aggregates is somehow determined and favored by the α-helical conformation of the peptide chains. In this case, the unordered form of LL-37 being prevalent in a salt-free Tris-HCl buffer (Figure 3) should induce no or completely different CD motifs. Interestingly, this is not the case since very similar induced exciton CEs were measured to those obtained in PBS solution (Figure 5). One possible reason for this result is that PQS binding promotes the disorder-to-helix conformational conversion of LL-37 as it has been observed previously with various pharmaceutical agents, food dyes, and porphyrin metabolites (Zsila et al., 2018; Zsila et al., 2019). By this way, the helical transformation of the unstructured peptide chains in association with the J-aggregation of PQS may explain the same induced CD pattern. Such a concerted mechanism, however, is unlikely due to the missing CD spectroscopic evidence of helical folding. Instead of development of the double dichroic minima characteristic of the α-helical conformation (Figure 3), only a slight red shift and intensity reduction of the main negative peak could be noticed (Figure 6). The negative CE evolved around 230 nm was also displayed in PBS (Figure 4), where the α-helical form of LL-37 is dominant. Therefore, it is not of a peptide origin but stems from an optically active electronic transition of PQS. The C-terminal of LL-37 is rich in disorder-promoting residues including Pro33, Glu36, and Ser37, suggesting the potential persistence of local disorder even under helix-promoting environmental settings. This prediction is in line with NMR studies made on LL-37 adsorbed on the surface of dodecylphosphocholine micelles (Porcelli et al., 2008). While the major portion of the peptide chain adopts helical conformation, the C-terminal residues from 30 through 37 are unstructured. The invariant conformational features of this segment may render it a suitable template to initiate a similar chiral supramolecular organization of PQS molecules both in PBS and in a salt-free Tris-HCl buffer.

Notwithstanding the above, some spectral differences in the CD curves recorded in PBS and Tris-HCl buffer should be noted. At the same PQS concentrations, three and two times more intense CEs were induced in PBS (cf. Figure 4 and Figure 5). This might be due to the more uniform conformer population of LL-37 present in PBS where the major fraction of the peptide chains is helical, having a short, unordered C-terminal tail. In this case, the chiral inductive effect of the C-terminal end prevails besides the neutral or supportive contribution of the α-helical segments. In contrast, the much more heterogeneous, disordered conformational ensembles of LL-37 characteristic of an anion-depleted medium impair the measure and purity of the supramolecular chiral ordering of J-aggregated PQS units.

To estimate the apparent dissociation constant of PQS–LL-37 interactions, absolute ellipticity values of the CD minimum at 230 nm were plotted against the increasing PQS concentrations achieved during the titrations (Supplementary Figure S2). A nonlinear regression analysis of the data points yielded 290 μM and 1.43 for the K _d and the Hill coefficient, respectively. The greater than 1 value of the Hill coefficient suggests positive binding cooperativity between the PQS molecules.

To monitor the PQS-provoked aggregation, dynamic light scattering measurements were performed in PBS solution. No particle formation was detected for the native peptide sample at 37 μM. Titration of LL-37 with PQS induced significant changes in the mean hydrodynamic diameter, resulting in large-sized aggregates (Supplementary Figure S3). Similarly, the shift in the correlation function toward higher decay times refers to the formation of larger assemblies in the micrometer range (Supplementary Figure S4).