A Survey of Gasoline Ameliorator, Methyl-Tert-Butyl Ether (MTBE) on Bovine Serum Albumin: A Spectroscopy and Molecular Dynamic Simulation Study

Background: Methyl-Tert-Butyl Ether (MTBE) as a gasoline modifier is frequently added to fuels and used in plenty of worldwide applications. MTBE biodegradation in groundwater occurs slowly and produces water miscibility; therefore, it causes diverse environmental and human health concerns. Objectives: The interaction of MTBE with bovine serum albumin (BSA) as a model protein at physiological conditions is investigated to illustrate the possible interactions of MTBE with the body’s proteins. Materials and Methods: Uv–visible, fluorescence, circular dichroism (CD) spectroscopy methods, and molecular modeling were used to analyze the MTBE’s effect on BSA structure and dynamics. The constant protein concentration and various MTBE contents were used for possible interactions. Results: The protein structural analysis shows that MTBE binds to BSA via positive enthalpy and entropy via hydrophobic interactions. Molecular docking shows the participation of several amino acids in the MTBE-BSA interaction. The CD spectroscopy results show that the BSA structure was not changed in the MTBE concentrations utilized in the study. Molecular dynamics (MD) simulation results suggest that MTBE can slightly change protein structure in the last 50ns. Conclusion: Comparing experimental and MD simulation results demonstrated that the BSA secondary structure was maintained in the low concentration of the MTBE. The entropy and enthalpy parameters asserted the hydrophobic interaction was the major force in the interaction between the BSA and MTBE.


Background
Methyl-tert-butyl ether is an organic compound with a chemical formulation of (CH 3 ) 3 COCH 3 . MTBE, also known as tert-butyl methyl ether, is a volatile, flammable, and colorless liquid. MTBE has been used as an oxygenating compound to improve gasoline octane levels since the late 1970s and replaced by the carcinogenic compounds of Tetraethyllead (CH 3 CH 2 ) 4 Pb. MTBE improves engine combustion efficiency while also lowering carbon monoxide, sulfur, ozone, and aromatic benzene emissions into the environment (1,2). Yearly, above 20 million tons of MTBE is manufactured and used globally (3). It is not surprising to show that MTBE by physicochemical properties such as high-water solubility, high volatility, and slow biodegradability found in the environment as a pollutant to enter atmosphere and groundwater via accidental leakage during production, transportation, and storage (4,5). Therefore, exposure to this compound via inhalation or drinking water affects human health. In 1999 the California Environmental Protection Agency (Sacramento, CA, USA) and the US Environmental Protection Agency (EPA) classified MTBE as a possible human carcinogenic agent (6). According to research, MTBE was shown to build in the bloodstream and breath, causing skin and eye irritation, diarrhea, vomiting, fever, cough, dizziness, tiredness, and headaches (7). Several studies confirmed that MTBE causes kidney and liver damage and makes leukemia and Leydig cells testicular tumors, especially in animals, mainly rodents (8)(9)(10)(11)(12). Despite many studies on cellular toxicity, few studies exist on MTBE's toxic effects at the molecular level or MTBE protein interactions. MTBE protein interactions (13,14). Proteins are the most important bioactive molecules in life; hence, a serum's reduction or rise in protein levels might be used as a key indicator for clinical diagnosis and health assessment. Furthermore, protein research with exogenous compounds such as contaminants is critical for a health assessment (15). After quickly entering the bloodstream, MTBE interacts with the blood proteins (16,17). One of the main soluble proteins in the circulatory system is serum albumin, with diverse physiological functions such as transporting many exogenous and endogenous molecules (18,19). The protein-ligand interactions and binding processes are widely studied using serum albumin as a model protein. Thus, regarding 76% structural homology with human serum albumin (HSA), bovine serum albumin was used extensively for protein-ligand interactions (20,21). BSA has three homolog domains; domain I, domain II and domain III, divided into nine loops (L1-L9) with 17 disulfide bonds. These loops formed a sequence of large-small-large loops and built a triplet in each domain. Each domain includes two subdomains, A and B. α-helix is a predominant BSA secondary structure (22).

Objectives
MTBE accumulation in the bloodstream and breath affects human health. As a result, the effects of MTBE on the structure of BSA were investigated using experimental methods such as fluorescence spectroscopy and circular dichroism (CD). The binding of MTBE to the protein was also shown using molecular dynamics (MD) modeling.

Materials
Bovine Serum Albumin (BSA) was purchased from Canada-Bioshop Company. Methyl-tert-butyl ether (MTBE) was prepared from ACROS organics. Phosphate Buffered Saline (PBS) was obtained from Bio Basic Company. All solutions were prepared with double-distilled water.

UV-Visible Spectroscopy
The UV-visible absorption spectra (200-700 nm) of BSA solution (2.25 μM) were performed in different concentrations of MTBE (0-9λ, each λ equal 8.3 μM), and it was recorded after zero and three-hour incubation at 37 °C. UV/Vis spectrometer Perkin Elmer measurements were carried out from 200 to 700 nm using a slit of 5 nm and a scan speed of 250 nm/min with jacketed cell holders. All experiments were run in phosphate-buffered saline containing 2.7 mM potassium chloride, 137 mM sodium chloride, and 10 mM phosphate buffer at pH 7.5 in a conventional quartz cell.

Fluorescence Spectroscopy
Intrinsic fluorescence was determined using 2.25 μM of BSA with a path length 1 cm cell at 37 °C. All fluorescence measurements were obtained with a fastscanning Cary Eclipse Agilent spectrofluorometer (Cary Eclipse, Varian, Australia). The emission spectra were analyzed between 300 and 440 nm, with an excitation wavelength of 280 nm. MTBA concentration was varied from 0-25λ (each λ equal 8.3 μM). The Stern-Volmer constant (K sv ) is evaluated to the quenching efficiency of MTBE. This parameter was calculated using the equation of F 0 /F= 1+ [MTBE]K sv , where F 0 and F are the emission intensities in the absence and the presence of the MTBE, respectively (23).

Circular Dichroism (CD) Spectroscopy
In the presence of MTBE, changes in the secondary structure of BSA were observed. CD spectroscopy measurements were performed in an Aviv 215 spectropolarimeter utilizing a 1 mm path length cell in μM) with different concentrations of MTBE 0-20λ concentrations (each λ equal 8.3 μM), at 37 °C. Far UV-CD measurements of samples are performed using Aviv, Model 215, and USA spectrometer. The spectra were recorded in a 1 mm quartz cell and step size of 1 nm with three or two accumulations.

Thermodynamics Parameters
The fluorescence emission spectra of the samples were examined at two different temperatures (298.15 and 310.15 K) to determine the thermodynamic parameters (ΔH, ΔS, ΔG). The van't Hoff equation was used to compute ΔH: K b(T2) and K b(T1) are the binding constants at the first and second temperatures, respectively, and R is the universal gas constant (8.3145 j/k.mol). The thermodynamic equation was used to calculate ΔG and ΔS:

Molecular Dynamics Simulation
The initial coordinates of BSA were obtained from its crystal structure (PDB ID: 3VO3) (24), and the MTBE structure was taken from PubChem CID:15413 (http:// pubchem.ncbi.nlm.nih.gov/) (Fig. 1). MD simulations were performed with GROMACS, version 5.1.2 simulation package using the GROMOS 96-54a7 force field. Water molecules of the crystal structure were removed. The BSA was simulated in pure water. BSA and BSA-MTBE complex were immersed in a 90000-molecule cubic box water (with dimensions 10×10×10 nm) using the simple point charge (SPC) water molecules. Chloride and sodium ions were used to neutralize the systems. Using the Berendsen method with 0.1 and 0.5 τps, the temperature and pressure were held near the target values (300 k and 1 bar, respectively) (25). The LINCS algorithm was used to constrain the length of covalent bonds (26). A short-range spherical cut-off 1 nm was applied for all nonbonded interactions. The Particle Mesh Ewald (PME) approach was used to calculate the long-range interactions (27). The steepest descent approach was employed to reduce the energy consumption of the MD simulation systems. The conventional (NVT) ensemble was run for 0.5 ns after energy reduction. All systems were then equilibrated using an isothermalisobaric (NPT) for 1 ns. The simulation length for each BSA-MTBE and free BSA system was 200 ns (28). Given that the allowable amount for MTBE is 15 μg maximum per liter by Iranian national standard and 40 μg per liter for the US, the necessary calculations were performed, and it was found that for each box of 90000-water molecules, a much smaller number than one MTBE molecule was obtained. Since this number was not mathematically practicable, one molecule was selected, equivalent to a concentration of 616 μM (54.3 mg. L -1 ) in the box. The BSA concentration should be 41.7 g. L -1 is performed. Due to this study's specific conditions, the size of the protein and the smallness of the ligand selected were much greater than that of the in vitro concentration. However, this selective amount of simulation was also performed in the laboratory phase to make a reasonable comparison. The minimization was completed when the root-mean-square deviation (RMSD) achieved a maximum value of 0.30. The binding mechanism of MTBE with bovine serum albumin was investigated using molecular docking. Simulations of free BSA and BSA-MTBE were carried out.

Molecular Docking
In this work, the AutoDock software (Version 4.2.6) was utilized to simulate MTBE's binding site upon interactions with BSA. In general, the Lamarckian genetic algorithm (LGA) was applied for the local search (Solis and Wets method). Kollman partial charges and polar hydrogens were added to the BSA structure during the simulation. Then in the AutoDock tools package, the partial atomic charges were calculated using the Gasteiger-Marsili method, and after merging non-polar hydrogens, rotatable bonds were assigned (29). The number of grid points in the x, y, and z axes was 126 Å ×126 Å ×126 Å. The distance between the two connecting grid points was 0.375 Å (roughly a quarter of a carbon-carbon single bond). A structure with the lowest energy content and the best binding site was selected. Using discovery studio and ligand scout software, hydrogen bonds and hydrophobic interaction sites were assessed with their binding partner.

UV-vis Absorption Spectra Investigation
BSA is composed of 585 amino acid residues, and it has several aromatic amino acids. BSA has two absorption bands at near 220 nm, which is related to α-helix structure and represents the conformation of BSA, and 280 nm, which is linked to the π-π* transition of the aromatic residues such as Trp, Tyr, and Phe residues (30,31). The UV-visible spectra of BSA solutions in the presence of MTBE are shown in Figures 2A and  B. Electronic absorption spectroscopy is an impressive method to analyze the binding mode of protein and small molecules. To record the possibility of MTBE interaction with BSA, spectroscopic titration of different concentrations of MTBE with a constant BSA concentration has been performed. The result shows that MTBE could affect the BSA structure immediately (Fig. 2A). The association remains the same after a 3-hour incubation period (Fig. 2B). The intensity of the two bands has risen, indicating that the hydrophobicity around aromatic residues has decreased and BSA's -helical content has grown. Consequently, our findings suggest that MTBE may affect the microenvironment of aromatic acid residues in BSA.

Intrinsic Fluorescence Study
Fluorescence spectroscopy is a high-sensitivity technique for the protein's structural transition in interaction with different ligands (32). BSA consists of several aromatic residues that contribute to the BSA intrinsic fluorescence absorption (Trp, Tyr, Phe) (33). Two tryptophan residues, Trp-134, located on the surface of subdomain IB, and Trp-212, located within the hydrophobic binding pocket of subdomain IIA, possess intrinsic fluorescence in BSA (21,34). The fluorescence emission spectrum of BSA in the presence of various concentrations of MTBE is given in Figure 2C. BSA has a strong fluorescence emission peak approximately at 350 nm. It was discovered that the emission intensity of BSA increases at low concentrations and decreases somewhat with rising MTBE concentrations, with no discernible emission wavelength change. This trend represents the decreasing BSA intrinsic fluorescence owing to interaction with MTBE. The solution property changed in terms of increasing MTBE concentrations. The same alternation caused the micro-environment around residues to have intrinsic fluorescence, and residue with innate fluorescence intensity could fluctuate freely. Fluorescence emission decreased owing to this increased residue moving. The Stern-Volmer plot showed in Figure 2D. The

D) C)
Stern-Volmer plot of BSA-MTBE illustrates that the quenching of interaction of BSA by MTBE is in good agreement (R 2 = 0.91). The Stern-Volmer constant was determined from the slope of the linear curve and equaled 0.6 mM -1 . These results in a decrease in the fluorescence intensity, and the high K sv value of the MTBE shows that MTBE could interact with BSA. The thermodynamic characteristics of the BSA and MTBE interaction were determined at two temperatures, 298.15 and 310.15 K ( Table 1). Considering ΔH and ΔS are more significant than zero, it is concluded that hydrophobic interactions are the primary driving forces for the BSA-MTBE binding.

Circular Dichroism Measurement
CD spectroscopy analysis is used to accurately evaluate the secondary structural changes of BSA under different MTBE concentrations. Far UV-CD spectra of BSA in the absence and presence of MTBE are given in Figure 3. For α-helix-rich protein like BSA, CD spectra show two negative peaks at 208 and 222 nm (35). Studies reported that native BSA has around 65% of α-helical secondary structure content (23). The CD results indicated that MTBE increases the content of the secondary structure in low concentrations (Table 2, Fig. 3). Therefore, by increasing the MTBE concentration, the secondary structure content is decreased.    This, like fluorescence results, shows that at low concentrations, MTBE increases the structural stability and has a different effect at higher concentrations.

Molecular Dynamics Analysis
MD modeling has the potential to reveal minute information regarding protein-small molecule interactions. MD data might reveal structural alterations and system stability that a protein may experience in the presence of tiny compounds (36). To investigate the conformational dynamics of MTBE interaction with BSA, 200 ns long MD simulations were done at a temperature of 300 K. The structural stability and energetic features of BSA-MTBE interaction were monitored based on the time course of the C α rootmean-square deviation (RMSD) concerning to their initial conformation. The RMSD values for free BSA and BSA-MTBE complexes during MD simulations are shown in Figure 4A. The RMSD of BSA-MTBE is almost stable and fluctuates between 0.4 and 0.7 nm. The fluctuation of BSA-MTBE is approximately more stable after 100 ns. However, free BSA fluctuates between 0.3 nm and 0.6 nm with a slightly variable pattern throughout the 200 ns simulation. The RMSD value of free BSA represents different RMSD values than the BSA-MTBE complex; therefore, these results indicate that the conformational change occurred during the BSA-MTBE interaction. The root-mean- square fluctuations (RMSF) of protein backbone atoms were determined for the free BSA and the BSA-MTBE complex. During MD trajectories, the RMSF calculates the average atomic mobility. The more flexible residues of protein structure show higher RMSF values (37). As shown in Figure 4B, clearly different flexibilities in the binding sites of BSA in the presence and absence of MTBE are observed. The binding sites in subdomains IA and IB are more flexible when MTBE interacts with BSA. In the lack of MTBE, IIB, IIIA, and IIIB subdomains are more flexible. This pattern shows that the structural stability of BSA alters during the MTBE interaction. Figure 4C shows the radius gyration of both free BSA and BSA-MTBE. The compactness of a protein can be determined through the radius of gyration (R g ), which expressed that the lesser the R g more compact the molecule (38). The lower R g result indicates that BSA-MTBE has a compact structure throughout MD simulation. The stabilization of the BSA-MTBE complex is illustrated in the consistency of R g values after 30 ns. Moreover, solvent accessibility surface area (SASA) for the proteins was also measured and depicted in Figure 4D. The decreasing SASA values represent relative compactness. The lower SASA result indicates that BSA-MTBE has a compact structure throughout MD simulation and obtains from R g and RMSF.

Molecular Docking Results
To investigate the binding site of BSA to MTBE, molecular docking was performed. The low energy binding site with the best fits MTBE is shown in Figure  5. The docking result indicates that a binding site on BSA has a perfect binding affinity for MTBE; the following residues interact with MTBE: Val40, Lys132, Lys131, Phe36, Trp134, Lys20, Asn44, and Val34 are located in IA and IB subdomains (Fig. 5A, 5B). MTBE binds to BSA with a binding energy of -6.3 kcal/mol. More specifically, subdomain IA and IB have more RMSF values. It seems that this fluctuation is related to MTBE interaction with this region of BSA. Trp134 is involved in MTBE interaction hence decrease in fluorescence intensity occurs during these interactions and could affect the microenvironment polarity of Trp134 in subdomain IB. According to molecular docking and MD modeling, the hydrophobic head of MTBE (methyl) interacts with the hydrophobic cavity of IA and IB of the BSA protein, causing a structural alteration in BSA. Compared to free BSA, MTBE-BSA is more stable and has a compact structure.

Discussion
In order to improve air quality, the production and use of fuel oxygenate as MTBE has increased since the early 1990s. The prevalent use of MTBE in gasoline has been accompanied by widespread release into the environment. MTBE is a comparatively unreactive compound in the environment (39). MTBE can pollute the environment, quickly enter the blood, and significantly affect the proteins. The previous study showed that MTBE interacts with hemoglobin, perturbs its structure and function, and degrades heme; therefore, adversely affects oxygen affinity and transportation (17). Besides, MTBE could alter insulin structure, form insulin aggregation, and generate reactive oxygen species (14). The in vivo studies illustrated that MTBE has genotoxic potential and forms adducts with DNA (40). Serum albumins are a key component of blood plasma because they aid in the binding and transporting of a wide range of exogenous and endogenous chemicals, such as hormones, fatty acids, and medicines (22). As a result, concerns about potential inhalation health effects and, more recently, increased concerns about water contamination of the side effects of this environmental pollutant on human health were investigated.

Conclusion
The fluorescence spectroscopy results show that interaction between widespread environmental pollutant MTBE and BSA decreases fluorescence emission intensity in higher concentrations. Furthermore, UV-vis and CD results indicate the binding interaction of MTBE with BSA by an increase in α-helix of the secondary structure in low concentrations. At the MTBA doses utilized in this experiment, compression occurs in the protein structure. The hydrophobic interaction was the primary force between MTBE-BSA complexes, according to the thermodynamic characteristic. MD simulation and docking consequences confirmed experimental results. Our result demonstrated that the BSA secondary structure is maintained in low MTBE concentration; however, it's detrimental in higher concentrations.