AA was purchased from Sigma-Aldrich; Merck KGaA. The stock solution was prepared in buffer (KH₂PO₄=0.925 gr, KHPO₄=0.825 gr, 169 ml with double-distilled water, pH=6.8). HSA was purchased from Sigma-Aldrich; Merck KGaA. The HSA solutions were prepared 30 min prior to the experiments. In the present study, 40 µM HSA and 10 µM AA concentrations were used. For denaturation experiments, 6 M urea was prepared.

To evaluate the effects of AA on protein chemical stability, the HSA chemical denaturation profiles were recorded by the titration of 40 µM protein solution with aliquots from a 6 M urea stock solution. These experiments were performed in the absence and presence of a 10 µM AA concentration. The protein conformational changes were obtained at wavelength of 280 nm by spectrophotometric technique.

HSA thermal denaturation fluorescence measurements were performed using a spectrofluorometer (Cary Eclipse model 100, equipped with a thermostatically controlled cuvette compartment). Variable temperatures were utilized (10-90˚C) and the emission spectra were recorded at 357 nm with excitation wavelength of 290 nm with an increase of 1 nm. The HSA (40 µM) intrinsic fluorescence was evaluated using thermal scanning, in the absence and presence of 10 µM AA.

The HSA intrinsic fluorescence evaluations were performed in the presence and absence of AA via a spectrofluorometer (Cary model Eclipse) using a 1 cm quartz cell and a thermostat bath. A 40 µM HSA solution was prepared and titrated with increasing concentrations of AA (0, 40, 80, 120, 160, 200, 240, 280, 320 and 360 µM) in 50 mM buffer solution with 298 K temperature. An appropriate blank which corresponded to the buffer was subtracted in order to correct for the background fluorescence. The excitation wavelength was 290 nm, and the emission spectra were recorded from 300 to 420 nm. The maximum emission intensities were used to calculate the binding constants, binding sites occupation and thermodynamic parameters evaluation (16). The quenching process is described by the Stern-Volmer equation: F₀/F = 1 + K_SV[Q] = 1 + k_qτ₀[Q]. Where F₀ and F are the steady-state fluorescence intensities in the absence and in the presence of quencher (AA), respectively, K_SV the Stern-Volmer quenching constant, and [Q] the concentration of the quencher. kq is the quenching rate constant of the biomolecule, τ₀ is the average lifetime of the biomolecule without quencher (17).

The number of binding sites and the binding constant of the AA and HSA interaction were obtained by the following equation: log[(F₀-F)/F] = log(K_b) + n log([Q]), where K is the binding constant for a site and n is the number of bindings per albumin, respectively. The plot of log [(F₀-F)/F₀] versus log [Q] yields log(K_b) as the intercept and n as the slope. The values for K and n were obtained from the intercept and the slope (18). In order to map the HSA and AA interactions, the thermodynamic parameters were calculated using the Vant Hoff equation (19,20): ln(K_b) = -(ΔΗ/RT) + (ΔS/R). Where the K_b is the binding constant, R is the universal gas constant (8.314 kJ/mol), ΔH and ΔS are enthalpy and entropy changes during the quenching process. The free energy changes (ΔG) associated with the interaction of AA and HSA were calculated from the following equation: ΔG = ΔH - TΔS = -RT ln(K).

Fluorescence lifetime measurements were performed using an Edinburgh Instruments (FLS 920) spectrofluorimeter in laser mode with excitation and emission wavelengths of 255 and 320 nm, respectively. The spectrofluorimeter in photon counting mode equipped with a temperature-controlled cell connected to a circulating water bath was used. The fluorescence lifetime of 40 µM HSA in buffer phosphate (pH 7) was measured in the presence and absence of 100-fold concentrations of 0, 20 and 80 µM AA. Using a double-exponential decay function of the decay profiles with F900 analysis software, the lifetime values were determined from the reconvolution fit analysis. Applying the reduced χ² value, the goodness of fit was assessed (close to 1 in all cases).

Furthermore, according to the following equation and using the amplitude β_i with the Euler angle in the i^th frame, component lifetime τFi, the mean fluorescence lifetime (τF) was calculated by taking into account the amplitude α_i (21): τ_F = Σ_i (β_i τ_Fi²)/Σ_i (α_i τ_Fi).

CD measurements were performed using a JASCO spectropolarimeter (model J-800; JASCO Applied Sciences) equipped with a thermoelectrically controlled cell holder under a constant nitrogen flow. Cuvettes with path lengths of 1 mm were utilized. Far-ultraviolet (UV)-CD spectra were recorded in the range of 190-260 nm. Near-UV-CD of the adjacent chain of aromatic amino acids was absorbed in the range of 250-290 nm. A concentration of 40 µM HSA and AA concentrations of 0, 20, 80 and 100 µM were used. The slit width was set at 5 nm, and the speed of scanning was 30 nm/min^-1.

Fluorescence anisotropy is a phenomenon where the light emitted by a fluorophore has unequal intensities along different axes of polarization. The steady state anisotropy (r) is given by:

where IVV and IVH are the intensities measured with vertically polarized excitation, as indicated by the first subscript, and detected through vertically or horizontally oriented emission polarizers, respectively, as indicated by the second subscript. The factor

which is measured using horizontally polarized excitation, corrects for instrument polarization bias. Fluorescence spectra and steady-state emission anisotropies of all HSA samples were measured using a Cary Eclipse fluorimeter (Agilent Technologies, Inc.). Anisotropy was measured with a manual polarizer and the G factor correction was performed manually, with a reference fluorophore. The labelled protein was then excited at start (635 nm) and stop (640 nm) points. Furthermore, the parallel and perpendicular emissions were monitored at maximum emission of 580 nm with a manual polarizer. The anisotropy measurement of the protein was then repeated with AA (21,22).

To study the interactions between HSA and AA, molecular docking was performed by the Auto Dock Vina 1.5.6 program). The docking process was performed to predict the preferred orientation and binding affinity of the ligands. The HSA crystal structure was obtained from a protein data bank (4L9Q PDB code). The graphical images obtained represent the best pose which were prepared using LigPlot v.4.5.3 software (23).

The AA molecular docking process to HSA was performed using AutoDock Vina. The docking method in which receptor side chains and the ligand are flexible was used for the aforementioned process. To assess the permissible torsions for the ligand, characterizing the search space coordinates and adding polar hydrogen atoms to the protein, the graphical AutoDock tool was applied (24). Following this, the docking process of domain I and II was performed via a grid size of 126 x 126 x 126 along the X, Y and Z axes with 1 Å spacing. The lowermost binding energy for the docking conformation of AA2-HSA and AA3-HSA complexes was created using AutoDock Vina, which were assumed as the primary conformation for the MD simulation process.

The conformational modifications of ligand-enzyme complexes were estimated using a MD method via the GROMACS 4.5.4 (University of Groningen Royal Institute of Technology) package software and the GROMOS96 43a1 force field. A periodic box full of water molecules was designated for the complexes which was achieved by the MD method (25). The HSA topology characteristics were created using the aforementioned GROMACS program. The AA topology parameters were created by means of the Dundee PRODRG server (26). The complex was immersed in a cubic box of extended simple point charge water molecules. The steepest descent method of 10,000 steps followed by the conjugation gradient method for 10,000 steps was utilized to minimize the energy in order to release incompatible contacts. The system equilibration phase constant substance amount, pressure and temperature (NPT) and constant substance amount, volume and temperature (NVT) as performed at 300 K for 200 psec followed by MD production run for 20 nsec. As described in previous studies (27,28), the atomic coordinates were registered every 2.0 psec during the MD simulation process. In this section parameters, including root mean square deviation (RMSD) and root mean square fluctuation (RMSF), were measured.

Chemical and thermal denaturation profiles were obtained from urea titration and thermal scanning in the absence and presence of AA in specific concentrations, which are depicted in Figs. 1 and 2. Each profile is a sigmoidal curve; thus, this process is described as a single denaturant-dependent step based on the two-step theory. Determination of the standard Gibbs free energy of denaturation (ΔG⁰), as a criterion of the conformational stability of a globular protein, is based on two state theory and the equation 1(29): ΔG⁰ = -RT ln(K). The secondary plots of Figs. 1 and 2 are illustrated insets. From these linear plots, the ΔG⁰ varies linearly in a denaturation-dependent manner (urea concentration and temperature) over a limited region. The equation for determining the ΔG⁰ is as follows: ΔG⁰ = ΔG⁰(H₂O) - m[denaturant]. Where ΔG⁰(H₂O) is the free energy of conformational stability in the absence of a denaturant, while m is the measure of ΔG⁰ dependence in denaturant concentration.

In chemical denaturation, [Urea]₁/₂ is the denaturant concentration which the protein needs to reach to halve its two-state transition. In thermal denaturation, the protein melting point (T_m) is a temperature which the protein needs to reach to halve its two-state transition. The magnitudes of ΔG⁰(H₂O), [Urea]₁/₂ and T_m were determined from replots, and are summarized in Table I.

Internal fluorescence spectroscopy is a method which is widely used to investigate the microenvironment of amino acid residues by measuring the emission wavelength shift (30). The HSA internal fluorescence spectra with various amounts of AA are presented in Fig. 3. The HSA fluorescence intensity regularly decreased with the addition of AA. Furthermore, it was demonstrated that fluorescence quenching occurred in the binding. Additionally, it implies that the interaction between AA and HSA may affect the Trp residue microenvironment. Thermodynamic parameters of the binding reaction, obtained from the Stern-Volmer curve, confirm the binding force. Therefore, the temperature-dependent thermodynamic parameters were analyzed to characterize the acting forces between AA and HSA (31). The results of the data sequential analysis using the Vant Hoff equation are illustrated in Table II. These were confirmed parameters by Fig. 4.

Based on the results of the fluorescence lifetime measurements, adding 20 and 80 µM AA to the protein solution resulted in no significant change in the fluorescence lifetime of HSA (Fig. 5). Therefore, changing the concentration AA over time does not change, which indicates that the interaction is static.

The far-UV-CD spectra of HSA exhibited two negative bands in the 208 nm and 222 nm regions, which represent the β-sheet and α-helical structure of the protein, respectively. The near-UV-CD method obtained the HSA tertiary regular structure values (32). When the AA concentration was gradually increased from 20 to 100 µM, the CD exhibited fundamental changes (Fig. 6A and B).

HSA was labeled with a fluorophore and the anisotropy of the labeled protein was measured prior to and following AA binding. The dye to protein ratio was low and the anisotropy of unlabeled protein was 0.28. When AA was added to the labeled protein, the anisotropy decreased to 0.26. Interactions between HSA and AA and the third structure are not stable. This may be attributed to conformational changes in HSA and the generation of another binding site upon interaction with AA, which are in agreement with our CD and anisotropy results (33).

To determine the interaction of AA with HSA specific binding sites, a molecular docking process was performed. The main result to note of the reaction is the binding position of AA with HSA. One previous study demonstrated which knowledge about the protein-ligand binding may be useful to understand the function and effectiveness of AA and HSA as potential therapeutic agents (34). The selective side-chain residue flexibility is a great option which is available on AutoDock Vina docking software (35). The aim of the present study was to make the protein-ligand interaction environment more pragmatic in order to decrease the computer processing time. The conformational modification of the receptor with regards to ligand selectivity demonstrate that receptor flexibility may have an essential function in computer drug design, which is evaluated in current study (36). To determine the structural changes, which are induced by ligand binding (with AA), the MD simulation for free HSA (subdomain II and III) and AA2-HSA/AA3-HSA complexes was performed and the results were consequently compared. Based on structural characteristics, the stability of the HSA protein was able to be studied during the simulation time. The evolution time of RMSD from the preliminary structure was calculated for three simulation runs. The RMSDs of Cα atoms, which are representative of two complexes, are demonstrated in Fig. 7. The free protein and the protein involved in the complex achieved the appropriate stability after 1 psec, which proved the equilibration of the system. The RMSD values of the HSA Cα-atoms and the complexes indicated a near mean equilibration and oscillation value for the whole system with maintained stability up to the end of the simulation. To obtain the flexibility data, RMSF was measured. To identify the flexible regions of the molecule, RMSFs of Cα atoms were produced and are illustrated in Fig. 8. The RMSF value for the free protein and the protein involved in the complex have similar trends as illustrated in Fig. 8, while residues 262-267 and 474-480 in subdomain IIA and IIIA have the highest RMSF values. Due to molecular movement restriction which was induced by the ligand, the free protein demonstrates a higher RMSF value compared with the protein involved in the complex. Following MD analysis, the AA-HSA complex in site I was formed by the hydrogen bonds of Lys195 and Arg218 and hydrophobic interactions, as indicated in Fig. 9A. Additionally, the AA-HSA complex was created by the hydrophobic interaction in site II, as illustrated in Fig. 9B. The optimal probable conformation was obtained with AA and HSA following docking and MD analysis. The molecular affinity of AA2 and AA3 with HSA was reported as -5.8 and -5.0 kcal/mol, respectively. These results demonstrated which AA binds forcefully to the large hydrophobic cavity available on HSA at subdomain ΙΙA of site Ι. Studies have confirmed that IIA and IIIA subdomains are suitable as target and initial drug binding sites for the docking process. Each experiment which evaluates the ligand-binding site next to drug-binding sites I/II may be useful in selecting the best docking simulation position. To measure the structural modifications caused by the ligand (AA) binding the MD simulation process for free HSA and IIA/IIIA subdomains, experiments were performed and the results consequently compared. At present, the results demonstrated that AA has the ability to bind effectively to the large hydrophobic cavity, which is available on HSA at subdomain ΙΙA of site Ι. The HSA binding site was evaluated to recognize the site containing residues.