DETECTION OF LYSOZYME AMYLOID FIBRILS USING TRIMETHINE CYANINE DYES: SPECTROSCOPIC AND MOLECULAR DOCKING STUDIES

Due to their unique photophysical and photochemical properties and high sensitivity to the beta-pleated motifs, cyanine dyes are increasingly used as molecular probes for the identification and characterization of amyloid fibrils in vitro and the visualization of amyloid inclusions in vivo. In the present study the spectroscopic and molecular docking techniques have been employed to evaluate the amyloid sensitivity and the mode of interaction between the trimethine cyanine dyes and the native (LzN) and fibrillar (LzF) lysozyme. It was found that the trimethine association with non-fibrilar and fibrillar forms of lysozyme is accompanied by the changes in the dye aggregation extent. The molecular docking studies indicate that: i) the trimethines form the most stable complexes with deep cleft of the native lysozyme; ii) the dye binding to non-fibrillar protein is governed by the hydrophobic interactions, π-stacking contacts between aromatic or cyclopentane ring of the cyanine and Trp in position 63 or 108 and hydrogen bonds between the OH-groups of the trimethines and acceptor atoms of Asp 101 (AK3-7) and Gln 57 (AK3-8) of LzN; iii) cyanine dyes form the energetically most favorable complexes with the groove Gly 2-Leu 4/Ser 8-Trp 10 of the lysozyme fibril core; iv) cyanines-LzF interaction is stabilised by hydrophobic contacts, π-stacking interaction and hydrogen bonds. The dyes AK3-7, AK3-5 and AK3-11 were selected as the most prospective amyloid probes.

. Structural formulas of the trimethine cyanine dyes

Preparation of working solutions
The stock solutions of the examined trimethine cyanine dyes were prepared immediately before measurements by dissolving the dyes in dimethyl sulfoxide. The concentrations of the dyes were determined spectrophotometrically, using their extinction coefficients [15]. The lysozyme stock solution (10 mg/ml) was prepared by dissolving the protein in 10 mM glycine buffer (pH 2.0). This solution was used as reference for non-aggregated protein (LzN). The amyloid fibrils of lysozyme were prepared by the protein incubation in 10 mM glycine buffer at pH 2 and 60°C for 14 days. The amyloid nature of the protein aggregates was confirmed by ThT assay and the transmission electron microscopy. Hereafter, the fibrillar lysozyme is designated as LzF.

Spectroscopic measurements
The steady-state fluorescence spectra were collected on a FL-6500 spectrofluorimeter (Perkin-Elmer Ltd., Beaconsfield, UK) at 20ºC with excitation wavelength 590 nm using the 10 mm path-length quartz cells. The excitation and emission slit widths were set at 10 nm. To record the fluorescence spectra of the cyanines in the dye-protein complexes, the appropriate amounts of the stock solutions of the non-aggregated or fibrillar lysozyme were added to each dye in 5 mM sodium phosphate buffer (pH 7.4).
The absorption spectra of the dyes were acquired on Shimadzu UV-2600 Spectrophotometer (Japan) at 25ºC using 10-mm path-length quartz cuvettes. To measure the absorption spectra of cyanine dyes in the aqueous phase or in the complexes with lysozyme, appropriate amounts of the stock dye solutions in DMSO were added either to 5 mM sodium phosphate buffer (pH 7.4) or directly to the working protein solutions and were incubated for one hour. The deconvolution of the dye absorption spectra was performed in the Origin 9.0 (OriginLab Corporation, Northampton, USA) using the log-normal asymmetric function (LN) [20]: where A denominates the absorbance, max A is the maximum absorbance, v is the wavenumber, c v is the peak position,  expresses the asymmetry of the function defined as: where min v and max v represent the wavenumber values at half-absorbance. The parameter a designates the limiting wavenumber: Molecular docking studies To define the most energetically favorable binding sites for the examined dyes on the lysozyme amyloid fibrils, the molecular docking studies were performed using the the AutoDock (version 4.2) incorporated in the PyRx software (version 0.8) [21]. To further characterize the nature of the dye-protein interactions, the protein-ligand interaction profiler PLIP was used [22]. The dye structures were built in MarvinSketch (version 18.10.0) and optimized in Avogadro (version 1.1.0) [23,24].The crystal structures of hen egg white lysozyme (PDB ID: 3A8Z) was taken from the Protein Data Bank. The lysozyme model fibril was built from the K-peptide, GILQINSRW (residues 54-62 of the wild-type protein), using the CreateFibril tool as described previously [25].

RESULTS AND DISCUSSION
Fluorescence spectra of trimethines in the unbound state and in the presence of the native and fibrillar lysozyme are presented in Figure 2. The photophysical properties of the examined cyanines in the buffer solution were examined previously and summarized in the Table 1. Specifically, the trimethines AK3-1, AK3-3, AK3-5 and AK3-7 exhibit a negligible fluorescence in buffer solution with the emission maxima located between 640 and 651 nm, depending on the dye chemical structure [15]. Besides, trimethines AK3-8 and AK3-11 are characterized by a negligible fluorescence intensity in buffer and do not show clear emission and excitation maxima [15]. The addition of the non-fibrillar lysozyme to the AK31, AK33, AK38 and AK3-11 in the buffer solution was accompanied by a significant enhancement of the dye fluorescence intensity along with a slight bathochromic shift (1-6 nm) of the excitation and emission maxima. In turn, the association of AK3-5 and AK3-7 with LzN was followed by a slight fluorescence increase coupled with a ~ 70-nm shift of the dye excitation maxima to the shorter wavelengths. Simultaneously, a ~ 15 nm bathochromic shift of the AK3-5 and AK3-7 emission maxima was observed in the presence of non-fibrillar lysozyme. As shown in Figure 2 and Table 1, the spectral response of the trimethine dyes (except AK3-8) to the fibrillar lysozyme lies in a strong increase of the dye fluorescence ( f I ) as opposed to that in buffer ( 0 I ) and in the presence of nonfibrillar protein ( n I ), with a magnitude of the fluorescence intensity increase depending on the dye chemical structure. The fluorescence maxima of the trimethines are shifted by approximately 9-15 nm towards longer wavelengths compared to those observed either in the nonaggregated proteins or in the buffer solution, as illustrated in Table 1.   By analogy with the insulin model protein, we determine the specificity of the examined trimethines also to the lysozyme amyloid fibrils. Therefore, we calculated the amyloid detection factor (ADF) ( Table 1) characterizing the ability of a dye to selectively detect the fibrillar state over its native structure relative to the background fluorescence of the dye in buffer [15,26]: It appeared that the trimethines under study (except AK3-8) are characterized by the positive ADF values in the presence of the fibrillar lysozyme, being indicative of their higher sensitivity to the fibrillar protein aggregates compared to the non-aggregated state. Although the enchanced fluorescence was observed for AK3-8 in the presence of LzF, the magnitude of fluorescence intensity increase was higher in the presence of monomeric lysozyme, as judged from the negative amyloid detection factor for this dye. In the presence of lysozyme fibrils the amyloid specificity was found to decrease in the row AK3-7 → AK3-11→ AK3-5 → AK3-1 → AK3-3→ AK3-8. It appeared that AK3-7, possessing the lowest sensitivity to the insulin amyloid fibrils [15], has demonstrated the largest ADF value in the presence of the lysozyme amyloid fibrils. The relatively high amyloid detection factors were observed in the presence of lysozyme for AK3-11 and AK3-5 dyes. Remarkably, these dyes were highly sensitive also to the insulin amyloid fibrils [15]. However, the ADF values of the trimethines were found to be significantly higher in the presence of insulin amyloid fibrils (ADF values exceeded 15 for all cyanines except AK3-7 in the presence of insulin fibrils [15]) compared to lysozyme, suggesting a sensitivity of the examined cyanines to the fibril morphology.
To interpret the observed fluorescence responses and the binding data of the cyanine dyes, in the following studies we analyzed their absorption spectra free in buffer solution as well as in the presence of control and fibrillar protein ( Figure 3). For a more detailed analysis (by analogy with the data obtained for the insulin fibrils), we performed the decomposition of these absorption spectra using the log-normal asymmetric function (Eq. 1) [20].
The absorption spectra of AK3-5 and AK3-3 in buffer solution were represented as a sum of two separate bands, with a short-wavelength and long-wavelength spectral components corresponding to the dimeric and monomeric dye species, respectively. The corresponding absorption spectra of AK3-1, AK3-7, AK3-8 and AK3-11 in aqueous phase were deconvoluted into three bands representing the dye monomers, dimers and higher order aggregates (Figure 4). The deconvolution of the absorption spectra allowed us to calculate a set of parameters, viz.: i) the amplitude max A ( Table 2): i) the peak position c  , related to the environmental polarity [20] (Table 3); ii) the full width at half-maximum of the band (FWHM) ( Table 4); and iii) the peak asymmetry parameter  (Table 5).  I  40000  97000  50000  84168  20739  15353  II  8500  35171  33680  39000  8816  9711  III  14000  --8000  27500  18934  LzN  I  43112  22100  70503  50720  27768  14355  II  11652  9935  52474  52102  24562  5360  III  7895  11434  -2224  9627  30784  LzF  I  52327  32383  74203  46437  44261  53443  II  13891  6468  60928  54887  25834  22866  III  4918 1757 -39 2091 7296 The incubation of АК3-1, АК3-5 та АК3-8 (Table 2) with the non-fibrillar lysozyme resulted in the following changes of the amplitude of the bands in comparison with those observed in the buffer solution: i) the rise in the amplitude of the bands I (a 1.1-fold, 1.4-fold and 1.3-fold increase) and II (a 1.4-fold, 1.6-fold and 2.8-fold increase); ii) the drop of the amplitude of the band III for АК3-1 (1.8 times) та АК3-8 (2.9 times) and the band narrowing; iii) a 3.3-fold, 1.7-fold and 1.1-fold decrease in the amplitude of the band I for АК3-3, АК3-7 and АК3-11, respectively; iv) the rise in the amplitude of the bands II (AK3-7) and band III (AK3-11). It should be noted that the absorption spectra of AK3-3 in the non-fibrillar lysozyme were deconvoluted into three separate bands, where for the short-(dimeric dye form) and longwavelength (monomeric dyes form) bands the 3.5-fold and 4.4-fold drop of the amplitude was observed. Likewise, the incubation of AK3-3 with the non-fibrillar lysozyme resulted in the appearance of the III short-wavelength band of H-aggregates, the intensity of which even exceeded the amplitude of the band II.

EEJP. 4 (2022)
Olga Zhytniakivska, Uliana Tarabara, et al Furthermore, while comparing the с  values (Table 3), it can be seen that monomeric lysozyme produced a hypsochromic shift of the monomer band I (AK3-3), dimer band II (all dyes except AK3-1), and the H-aggregates band III (АК3-1, АК3-7 and АК3-8). In turn, the position of the band I for АК3-8 та АК3-11 shifted to the higher wavenumbers. Moreover, the incubation of АК3-1, АК3-5 та АК3-8 with the LzN resulted in the increase of the asymmetry parameter (Table 5) and the full width at half-maximum (Table 4) of the band II (АК3-1 and АК3-7). In the meantime, the presence of the non-fibrillar lysozyme led to the decrease of the full width at half-maximum of the band I (Table 4). Taken together, we can conclude that the trimethine dyes AK3-1, AK3 5, AK3-7 and AK3-8 bind to the native lysozyme mainly in the form of monomers and dimers, while AK3-3 and AK3-11 show a tendency to aggregate in the presence of the non-fibrillar lysozyme.

CONCLUSIONS
To summarize, in the present study the optical spectroscopy and molecular docking techniques were used to investigate the interactions between the trimethine cyanine dyes and lysozyme in the non-fibrillar and amyloid states. It was found that all cyanines under study are capable of distinguishing between the non-aggregated and fibrillar protein forms. The dyes AK3-7, AK3-5 and AK3-11 appeared to possess the highest amyloid sensitivity due to: i) significant fluorescence enhancement in the presence of LzF produced by the binding of the dye monomers to fibrillar lysozyme; ii) the lower binding affinity of the dye monomers to the nonfibrillar lysozyme in comparison with the aggregated protein.
In turn, significantly lower ADF values observed for AK3-3, AK3-1 and AK3-8 were interpreted as arising from the binding of dye monomers to the nonfibrillar lysozyme. The fluorescence response of cyanines in the presence of lysozyme fibrils led us to conclude that the trimethine dyes under study are sensitive to the fibril morphology.