3.1. Fluorescence measurement
Typically, fluorescence quench is categorized into three types: static, dynamic, and mixed. To investigate the fluorescence quench mechanism of AR to Hb and Mb binding, calculating the constant of fluorescence quench produced at different temperatures and analyzing it together with the experimental results of fluorescence lifetime are necessary (Figs. 1b, d). The fluorescence intensities of Hb/Mb are regularly measured and extinguished with the increase of the concentration of the added AR, which indicates that the AR interacts with Hb/Mb. Although AR appears at a fluorescence peak of 500 nm (not depicted in the figure), its maximum emission wavelength differs from that of Hb/Mb (near 328 nm versus 340 nm). Figure 1 also shows a redshift and a blueshift of the maximum emission wavelengths of Hb and Mb. Considering that the same phenomena can occur for proteins at low concentrations, this change cannot be taken as amino acid residue microenvironment changes(Tang et al. 2022). The Stern–Volmer equation was used to calculate the quenching constants at three temperatures (Lakowicz 1999).
$$\frac{{F}_{0}}{F}=1 + {K}_{sv}\left[Q\right]$$
3
.
where F0 and F denote the fluorescence intensity in the presence and absence of AR, respectively. Ksv refers to the Stern–Volmer quenching constant, and [Q] represents the concentration of AR. The obtained data were fitted to the Stern–Volmer equation to obtain the Ksv values at different temperatures (Table 1). The Ksv value of AR binding to Hb were 0.74 ± 0.04 × 104, 0.51 ± 0.02 × 104, and 0.44 ± 0.04 × 104. The Ksv value of AR binding to Mb are 1.38 ± 0.03 × 104, 1.28 ± 0.04 × 104, and 1.00 ± 0.04 × 104 L/mol. The binding constants (Ka) were determined utilizing the Stern–Volmer equation and the double-log plot method (Table 1). The Ka value of AR interaction with Hb is greater than that of AR interaction with Mb, indicating a stronger binding affinity between AR and Hb. This difference in Ka values indicates that the interaction between AR and Hb is tighter and more stable, which is consistent with the findings from RMSD analysis conducted during MD simulations. Moreover, the clustering table reveals that the n value is approximately 1, indicating that AR possesses a single binding site in Hb and Mb. The Ksv data in the table show that the trend of the quenching constants is opposite to the temperature variation, which suggests static quenching between AR to Hb/Mb.
Table 1
Binding constants, Stern-Volmer constant and thermodynamic parameters of AR and Hb/Mb at different temperatures
System | T(K) | Ksv×104(L/mol) | Ra | Ka×104(L/mol) | n | Rb | ΔG(kJ/mol) | ΔH(kJ/mol) | ΔS(kJ/mol) |
AR-Hb | 277 | 0.74±0.04b | 0.99 | 2.59±0.51c | 1.11±0.07 | 0.99 | -23.41 | -32.66 | -33.42 |
298 | 0.51±0.02a | 0.99 | 1.72±0.13b | 1.11±0.03 | 0.99 | -22.87 |
310 | 0.44±0.04a | 0.99 | 0.55±0.10a | 1.02±0.05 | 0.99 | -22.27 |
AR-Mb | 277 | 1.38±0.03b | 0.99 | 1.05±0.12c | 0.98±0.06 | 0.99 | -21.33 | -33.20 | -42.84 |
298 | 1.28±0.04b | 0.99 | 0.56±0.08b | 0.93±0.04 | 0.99 | -20.65 |
310 | 1.00±0.04a | 0.99 | 0.22±0.07a | 0.87±0.03 | 0.99 | -19.87 |
Ra and Rb denote the correlation coefficients of Ksv and Ka, respectively. |
The presence of different letters expresses significant differences (p < 0.05). |
Time-resolved fluorescence is an important method for probing the fluorescence lifetime of proteins, using which the effect of small molecules on the fluorescence lifetime of proteins can be observed as a means of determining the type of protein quenching. The formation of a complex between a protein and a small molecule that results in a quench of the protein is often referred to as a static quench, which generally does not result in a change in the fluorescence lifetime of the protein. Conversely, if there is a significant change in the fluorescence lifetime, then it is considered to be a dynamic quench or mixed quench(Wu et al. 2020). Figure 2 shows the results of the fluorescence lifetime, and the related data results can help us to determine the type of quench produced by the binding of AR to Hb/Mb. Stable fluorescence lifetime results are considered to be a static quench, whereas results with large variations in fluorescence lifetime are considered to be a dynamic quench(Hu et al. 2022).
$$\tau =\sum {\alpha }_{i}{\tau }_{i}$$
4
.
Where \({\alpha }_{i}\) is the pre-factor of the i-th term, \({\tau }_{i}\) is decay times, and τ is the average fluorescence lifetime.
The time-resolved fluorescence is shown in Table 2, and the average lifetime values of Hb and Mb are the same with the addition of different AR concentrations. With regard to the quality of fit, all the χ2 values are < 1.2, which indicates that the data are reliable. The average lifetime value of Free Hb and Free Mb was calculated to be 2.67 ns and 2.77 ns, respectively. The changes were minimal with the addition of AR, which may be due to the error of the measuring instrument, and the difference between their fluorescence lifetime is negligible. Based on the fluorescence lifetime results, the binding quench mode between them is a static quench.
Table 2
Fluorescence lifetimes of Hb and Mb with different concentrations of AR
System | τ1(ns) | τ2(ns) | τ3(ns) | α1 | α1 | α1 | τ(ns) | χ2 |
Blank Hb | 1.31±0.02 | 2.19±0.02 | 5.33±0.02 | 0.41±0.01 | 0.31±0.01 | 0.27±0.01 | 2.67±0.04 | 1.10 |
AR-Hb | 1.32±0.02 | 2.34±0.10 | 5.40±0.06 | 0.42±0.01 | 0.32±0.01 | 0.27±0.01 | 2.73±0.04 | 1.11 |
1.27±0.01 | 2.23±0.04 | 5.27±0.01 | 0.41±0.01 | 0.32±0.01 | 0.27±0.01 | 2.67±0.02 | 1.04 |
Blank Mb AR-Mb | 1.68±0.03 | 2.49±0.08 | 4.50±0.10 | 0.45±0.01 | 0.22±0.01 | 0.33±0.02 | 2.77±0.08 | 1.11 |
1.73±0.02 | 2.30±0.02 | 4.73±0.02 | 0.44±0.01 | 0.19±0.01 | 0.37±0.01 | 2.95±0.02 | 1.16 |
1.77±0.02 | 2.34±0.03 | 4.69± 0.03 | 0.44±0.01 | 0.20±0.01 | 0.36±0.01 | 2.91±0.03 | 1.12 |
3.2. Thermodynamic analysis
Based on the abovementioned data analysis, the binding quench mode of AR to Hb/Mb is static quench. The main interaction forces between AR to Hb and Mb can be characterized by the relevant thermodynamic parameters(Ross and Subramanian 1981).
$$In{K}_{a}= -\frac{\varDelta H}{RT}+ \frac{\varDelta S}{R}$$
5
.
$${\Delta }G= {\Delta }H- T{\Delta }S$$
6
.
T and R denote the thermodynamic temperature and gas constant (8.31 J mol− 1K− 1), respectively
Eq. (5) enables the determination of thermal changes (ΔH and ΔS) by fitting the Ka value to the corresponding temperature (T). Furthermore, Eq. (6) facilitates the calculation of the free energy change (ΔG) associated with binding.
In investigations involving the interaction, non-covalent forces, including van der Waals forces and hydrogen bonds, play prominent roles. The AR to Hb and Mb thermodynamic parameters can help us to understand the binding mode between them, and considering that A and B are negative, hydrogen bonding and van der Waals forces play a major role in the binding between proteins and small molecules(Hu et al. 2022). ΔG with negative values indicates that the process is spontaneous. Thus, the interaction of AR with Hb and Mb is spontaneous and dominated by van der Waals forces and hydrogen bonding.
3.3. Analysis of conformational impact
Synchronized fluorescence spectrometry boasts excellent selectivity, high sensitivity, and minimal interference, making it ideal for analyzing multi-component mixtures. Throughout the scanning procedure, excitation and emission wavelengths are maintained at a constant interval (Δλ = λem − λex). By setting the spectral interval to 15 and 60 nm, respectively, valuable microenvironmental details of Tyr and Trp residues can be extracted(Wu et al. 2021). Figure 4 (a, b, d, and e) illustrates the impact of varying concentrations of AR on the Tyr and Trp residues within Hb/Mb. Based on the figure, the fluorescence intensity of Trp of Hb and Mb is stronger than that of Tyr, which indicates that the fluorescence intensity of Hb and Mb by the addition of AR is mainly provided by Trp. In addition, the fluorescence peaks are blue-shifted or red-shifted when Δλ = 15 nm and Δλ = 60 nm, which indicates that AR has an effect on the environment around Trp and Tyr residues(Hu et al. 2022).
The CD spectra with and without different concentrations of AR are shown in Fig. 3(a, c), and the CD spectra allow us to study changes in the structure of Hb/Mb upon AR binding. Fluorescence peaks around 208 nm and 225 nm were evident in both CD spectra, primarily attributed to the presence of α-helix structures within the protein. The intensity of the fluorescence peaks at both locations decreased after the addition of different concentrations of AR, but the peak shapes did not produce changes. In Fig. 3. (b, d), predictions from MD simulations it is shown that natural Hb contains 74.07% α-helix, 6.01% β-turns, and 19.95% random coil. After the addition of AR, the content of α-helix decreases, and the content of β-turns and Random coil are both increased. Natural Mb contains 76.63% of α-helix, 7.43% of β-turns, and 17.16% of random coil, and after the addition of AR, the content of irregular curls increased, and the content of α-helix and β-turns decreased. The interaction between AR with Hb and Mb leads to protein unfolding and relaxation, which may have an effect on the hydrogen bonding in the α-helix to shift, resulting in a lower content of α-helix and a higher content of other secondary structures(Jia et al. 2019).
3.4. UV-visible spectrum
UV–visible absorption spectroscopy is a routine method for observing changes in protein conformation in the study of protein–ligand interactions (Cao et al. 2018). support the phenomenon of fluorescence quench produced by AR to Hb/Mb complexes, UV–visible spectroscopy was utilized to corroborate. The results are demonstrated in Fig. 4 (c and f), where it can be observed that in the absence of AR and with the gradual increase in the molar concentration of AR, two absorption peaks appeared around 280 nm versus 408 nm. The maximum absorption peak near 280 nm is a characteristic peak for many proteins, which are produced by the phenyl groups of Trp and Tyr residues (Fang et al. 2018). The Soret band of Hb and myoglobin produces an absorption peak near 410 nm (Mahato et al. 2010).
The absorption peaks of Hb/Mb at 280 and 410 nm increased with the gradual increase of AR concentration. However, the absorption peak of Mb at 280 nm has a significant blueshift, which may be due to the effect of AR on tryptophan and tyrosine in Mb, resulting in the change of the peak, but it also shows an elevated trend. This phenomenon indicates a complex formation between AR and Hb/Mb. Static quench, dynamic quench, and the combination of dynamic and static quench are the main types of quench, but dynamic quench does not change the absorption of UV–visible spectrum, whereas static quench leads to the change of absorption spectrum(Zhang et al. 2012). Therefore, the findings indicate that the interaction between AR and Hb/Mb involves static quenching, which is consistent with the fluorescence quenching observations. The Soret band is generated because of the ᴨ–ᴨ* transition within the heme group in Hb/Mb, which is embedded in the hydrophobic pockets of amino acids such as Val, Leu, Phe, and Trp (Kaur et al. 2023). A slight change in the absorption spectrum near 410 nm was observed, suggesting the interaction of AR with Hb and Mb.
3.5 Computer simulation
3.5.1 Molecular docking
Molecular docking can help us probe the interaction between AR to Hb and Mb and the conformational changes of the proteins, as well as simulate the optimal binding position of the small molecules to the proteins and the magnitude of the binding energy. In studying AR interaction with Hb and Mb at the molecular level, molecular docking was performed on the basis of the Lamarck genetic algorithm of YASARA(Guan et al. 2020), and the 3D and 2D plots of the best-docked structure after docking 100 times are shown in Fig. 5, which are generally considered to be the best-docked conformation at the lowest energy conformation. Hb is a tetramer with a hydrophobic cavity in the middle, while small-molecule AR is surrounded by a hydrophobic cavity, and the binding sites promoting Hb binding are listed. Within the cavity surrounding the Hb binding site, amino acid residues are enumerated to delineate their roles in facilitating interactions. AR interacts with VAL1, ASN131, LYS127, TRP36, ARG141, TYR140, SER138, THR137, PRO95, SER133, LYS99, ALA130, and THR134 and binds stably with various interaction forces, particularly van der Waals forces, with an optimal docking energy of 9.42 kcal/mol. The residue interaction forces between AR and proteins stabilize their conformations, with van der Waals forces playing a pivotal role in enhancing the stability of AR within the hydrophobic cavity of Hb. AR interacts with LYS45, SER92, LEU89, HIS64, VAL67, VAL68, HIS93, HIS97, ILE99, PHE43, LYS42, and ASP44 of Mb, with an optimal docking energy of 8.77 kcal/mol, where the main interaction forces are van der Waals forces and carbon–hydrogen bonding. The results of the AR to Hb and Mb interaction forces are thermodynamically calculated and consistent. The molecule–protein interaction can be expressed as the contribution of all internal forces and the sum of the contributions of all interaction forces.
The abovementioned data help us to analyze the binding mechanism of AR with Hb and Mb comparatively. Under the influence of interaction forces, AR binds better to Hb, maintaining overall conformational stability. The binding energy can reflect the possibility of binding between the receptor and the ligand; higher docking energy indicates a tighter and more stable structure; by comparing the binding energy, we found that the binding energy of AR with Hb is high, which indicates that the conformation of AR with Mb is not as stable as that of AR with Hb, and that AR has more influence on the conformation of Mb.
3.5.2 MD simulation
Further exploration of the stability of the AR to Hb and Mb was conducted through MD simulations. Analyzing RMSD and RG provides additional insights into the structural alterations of these complexes, with RMSD values reflecting fluctuations in the protein backbone over the course of the simulation. The results are shown in Fig. 6; the average value of RMSD for AR to Hb within 70 ns is 2.29 Å, whereas the average value of RMSD for free Hb is 2.85 Å. In addition, the average value of RMSD for AR to Mb is 2.10 Å, whereas the average value of RMSD for free Mb is 2.12 Å. As shown in Fig. 6 (a and c), Hb and Mb after adding AR after 20 ns are in a smooth state, whereas the free Hb and Mb are in a fluctuating state from 0–70 ns, indicating that AR can stabilize the conformation of both proteins. The Rg values can indicate the changes in the size and compactness of protein–small molecules after an interaction; when the Rg value increases, the system is swollen, otherwise it is more compact. The average Rg value of AR to Hb is 23.89 Å, whereas the average Rg value of free Hb is 24.05 Å; the average Rg value of AR to Mb is 15.64 Å, whereas the average Rg value of free Mb is 15.42 Å, which indicates that the addition of AR makes the overall Hb more compact, while the space of Mb expands, which may cause the difference in the binding energy of the two proteins.
As shown in Fig. 6 (b and d), the interaction of AR to Hb and Mb have more pronounced fluctuations, indicating that AR has an effect on the conformation of both protein complexes when the interaction is carried out. In addition, AR to Hb fluctuates within 20–50 ns, whereas AR to Mb fluctuates more within 10–40 ns, so the comparison of the two complexes is discussed every 10 ns. As shown in Fig. 7 (a, b, c, and d), between 20 and 50 ns, AR interacts with the Hb amino acid residues LEU100, LEU104, VAL96, ASN107, TYR34, and LYS99, which are always present, indicating that these amino acid residues are the main contributors to their interaction force. As shown in Fig. 6 (b), an upward trend is observed between 20 and 30 ns, during which the amino acid GLU becomes LYS and the number of amino acids is decreasing. After 30–50 ns, the Rg value is again in a decreasing state, and the number of amino acid residues involved in the interaction increases, primarily providing the hydrophobic interaction force. As shown in Fig. 7 (e, f, g, and h), between 10 and 40 ns, during which AR interacts with Mb, the amino acid residues PHE138, ILE107, GLN26, GLY65, GLY25, VAL68, HIS93, LEU104, and LEU29 were found to be consistently present, indicating that these amino acid residues are the major contributors to their interaction force. As shown in Fig. 6d, AR to Mb was found to be in the range of 10–30 ns at an Rg value of 15.6–15.8; the fluctuation range is small, and the change in the number of interacting amino acids is not significant. By contrast, a clear downward trend is observed from 30 to 40 ns, and the number of interacting amino acids increases. We found that the change in Rg value is closely related to the number of amino acids involved in the interaction; when the number of amino acids increases, the system expands, leading to an increase in Rg value and vice versa. However, there is a rising trend in AR to Hb between 20 and 30 ns, but the number of amino acids decreases, and GLU changes to LYS. This phenomenon may be due to the fact that the side chain of LYS has a longer carbon chain, which may lead to the expansion of the molecular conformation, thereby increasing the Rg values.