Interaction studies unveil potential binding sites on bovine serum albumin for gut metabolite trimethylamine n-oxide (TMAO)

Interaction studies unveil potential binding sites on bovine serum albumin for gut metabolite trimethylamine n-oxide (TMAO) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Interaction studies unveil potential binding sites on bovine serum albumin for gut metabolite trimethylamine n-oxide (TMAO) Awadhesh Kumar Verma, Payal Gulati, GBVS Lakshmi, Anand Mohan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5176166/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jan, 2025 Read the published version in BMC Chemistry → Version 1 posted 10 You are reading this latest preprint version Abstract Trimethylamine-N-oxide (TMAO) is the most studied gut microbiota-derived metabolite and its binding to serum albumin has huge significance to understand the underlying mechanisms in human health and diseases. The interaction between BSA and TMAO has not been well studied; the binding mechanism, preferred binding locations, and subsequent conformation of BSA are still unclear. In the present work, spectroscopic, photoluminescence, Fourier transform infrared contact angle, circular dichroism, density functional theory, molecular docking, and molecular dynamics simulation approaches were used to probe TMAO-BSA interaction and identifying putative binding sites for TMAO on BSA. Non-covalent interaction of TMAO with bovine serum albumin forming stable docking complex with binding score of -3.6 kcal/mol was revealed through docking and simulation study. The docking complex of TMAO-BSA was found stable through hydrogen bond and electrostatic, Vander waals interaction, and water bridges via amino acid residues. Hydrophilicity/Hydrophobicity of the TMAO-BSA was studied using contact angle (Ɵ) measurement. Present study may be helpful for making strategies for Chronic Kidney Disease and other disease treatments wherein interaction of TMAO with serum albumin matters. Also, pharmacodynamics and pharmacokinetics of TMAO, as well as its mechanism of binding to BSA, may be better understood with the help of this study's findings. TMAO bovine serum albumin in silico DFT docking spectroscopy Molecular Dynamics simulation mmgbsa Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The human gut is the primary source of Trimethylamine-N-oxide (TMAO). There, the gut microbiota breaks down food components into TMA, which is taken into the bloodstream via the intestinal mucosa. Hepatic flavin monooxygenases in the liver subsequently convert TMA into TMAO.[ 1 ],[ 2 ] As per recent research reports, circulating TMAO is becoming known to be associated with an increasing variety of other chronic conditions, such as cancer, type 2 diabetes mellitus (T2DM), and renal disease. (Fig. 1) The biological effects of TMAO vary among several animals and tissue types. Water-stressed organisms and tissues use TMAO as an organic osmolyte to preserve cell volume. The capacity of TMAO to modify the structure and activity of a broad class of physiologically significant molecules is one of its roles. TMAO plays a significant stabilizing role in nucleic acid and the folded state of proteins. Studies on the thermodynamic effects of TMAO on proteins have demonstrated that TMAO inhibits denaturation of proteins and mitigates the effects of heat and pressure.[ 3 ] The accumulation of TMAO by the kidneys of mammals is a response to the destabilizing effects of urea (as well as inorganic ions) on macromolecular structures, such as proteins and nucleic acids, and to the inhibition of urea on activities, including ligand binding.[ 4 ] Figure_1: Formation of TMAO from various precursor obtained from ingested foods and its implications in different diseases. The main extracellular protein of the circulatory system, serum albumin, makes up roughly 60% of all plasma proteins. [ 5 ] It transports various medications, fatty acids, thyroid hormones, and steroids and gut microbiota derived metabolites such as TMAO, TMA, indoxyl sulfate etc. [ 6 ] Because it is more readily available and less expensive, bovine serum albumin (BSA) is frequently used in place of human serum albumin (HSA) in laboratory investigations. Because of their 88% and 76% closeness in amino acid sequences, BSA and HSA have comparable binding capacities.[ 7 ] Numerous studies have been conducted to examine the binding between gut microbial metabolites and natural chemicals with serum albumin.[ 8 ]–[ 12 ] Because serum albumin is a drug's transport carrier and has a major impact on its bioavailability, research on the relationship between albumin and TMAO is crucial. The capacity of these gut metabolites to bind determines how successful they are as pharmacological agents. [ 13 ], [ 14 ] Although TMAO has been extensively documented for its actions and implication in human health and diseases, no scientific data about its interaction with serum albumin is available. The interaction of TMAO and proteins such as bovine serum albumin (BSA) can involve physical binding, changes in protein conformation, and potential functional effects. As the mechanism behind specific binding of TMAO with Bovine serum albumin (BSA) and the effect of TMAO on protein confirmation are not clear. Hence, in the present study, efforts have been made to study these aspects in detail. It is evident that conformational change in serum albumin is prompted by interaction with metabolites such as TMAO, drugs molecules and dyes having lower molecular weight, that significantly alter the albumin secondary and tertiary structure. [ 15 ] Therefore, binding of gut metabolite TMAO with serum albumin is very important in the field of biomedical research. This study investigated the binding between bovine serum albumin with TMAO. The binding relationship was investigated using multi-spectroscopic (UV-vis absorption, CD, fluorescence, FT-IR) and contact angle methods. In addition, molecular docking studies were performed to better visualize the binding mode with BSA. Study parameters included TMAO binding energies with BSA, complex formation, and binding site involvement. 2. Experimental Section 2.1 Reagents and Buffer Bovine Serum Albumin (BSA) in lyophilized form, Tetramethyl ethylene diamine, Sodium dodecyl sulfate (SDS), Acryl amide, N, N methyl acrylamide, Bromo phenol blue and Trimethylamine N-Oxide (TMAO) with high purity were procured from Sigma Aldrich. Dialysis membrane (Snake Skin ™ Dialyis Tubing, 10K MWtO,16mm) was procured from Thermo Scientific. Glacial acetic acid was procured from TM Media. Sodium acetate was procured from Merck. Tris base was procured from G Bioscience. Ammonium per sulfate and glycine was procured from HiMedia. Comassie brilliant blue dye methanol and ethanol were procured from SRL Ranbaxy. 0.2-micron filter was procured from MDI and 2ml syringe was procured form HMD. MilliQ (18.2 MΩ·cm @ 25°C) from the Millipore water purification system was used for preparation of all the solutions. Acetate buffer at pH 4.3 was used for TMAO-BSA conjugate preparation. 2.2 Instrumentation 2.2.1 UV-Vis Spectroscopy The UV–vis absorption spectra of BSA were taken in the absence and presence of TMAO using UV-2600, UV-VIS Spectro-photometer SHIMADZU (Shimadzu Corporation in Japan), through 1.0 cm quartz cells keeping slit width 5 nm and 600 nm/minute scan speed. The spectra were recorded in wavelength range between 200–600 nm. 2.2.2 PL Studies Fluorescence was measured with the help of Fluorescence spectrophotometer (Cary Eclipse, Model G9800A, Agilent Technology,) with fixed excitation as well as emission slit width (5 nm) and 600 nm/minute scan speed. A quartz cuvette (3.5 ml) with path length 10 mm was used. 2.2.3 FTIR To determine the functional groups changes, residing on the surfaces of bio-conjugates; FTIR spectrophotometer were used in ATR mode (PerkinElmer Spectrum 1). The spectra were obtained in between range of 4000 − 400 wavenumber, with scan speed of 32. The contact angle measurement was done using drop shape analyzer (KRUSS, Germany) to check the hydrophobicity/hydrophilicity of the TMAO-BSA conjugate on ELISA plate. 2.2.4 CD Spectroscopy CD spectroscopic study was performed monitor the effect of TMAO on secondary and tertiary structure of BSA in acetate buffer solution. CD spectra were obtained in the UV region using JASCO-1500 CD spectropolarimeter associated with a Peltier temperature controller (PTC-517). Prior to the measurement, instrument was calibrated routinely with D-10 camphor-sulfonic acid under N 2 purging in lamp compartment, optics and sample chamber in a ratio of 1:3:1 to remove oxygen content and create the inert atmosphere. CD spectra i.e., ellipticity of the acetate buffer solution was recorded as a reference/background in the UV range from 190–250 nm using a cell (path length 1 mm) at room temperature (25°C), 1 nm bandwidth. The average of three measurements were displayed as final spectra with mean time 1 nm/sec. 2.3 Conjugate Preparation TMAO (in acetate buffer, PH = 4.3) was conjugated with BSA (acetate buffer, pH = 4.3) in different molar ratio’s including 1:1, 1:5, 1:10, 1:25, 1:50, 1;75, 1:100. A 3.5 mM of TMAO was mixed with 2 µM of BSA and allowed to react for 2 h at room temperature and for 72 h at 4°C. At the end of the reaction, dialysis was performed for 72 h with change of acetate buffer (pH = 4.3) after every 12 h. 2.4 Computational Details 2.4.1 Molecular Modeling and optimization of TMAO using DFT We have theoretically modeled 3D structure of gut metabolite TMAO using Marvin sketch software. Every time 2D and 3D cleaning was performed and 3D structural confirmation of the molecule was checked by visualizing them in Marvin view. Further, we calculated each and every parameter like atomic coordinates (XYZ), bond length, and bond angle using ChemBioDraw Ultra software and optimized the structure up to transition state followed by energy minimization using MM2. The 3D structures were optimized to transition state, and the energy was minimized sequentially using further optimization followed by energy minimization using Chemdraw and Chem3DPro. The Final optimization was done via DFT approach with Gaussian 09 software with standard basis set: 6-311G and RB3LYP functional. This optimized structure was further utilized for docking to monitor the interactions between TMAO and BSA molecule. [ 16 ] 2.4.2 Preparation of Receptor molecule BSA structure (Source organism: Bos Taurus , resolution: 2.70 Å and having 583 amino acid residues) was retrieved from Protein Data Bank having RCSB PDB ID: 3V03. The structure was cleaned of crystallographic water molecules and other crystalizing agents. Docking was performed to find the binding affinity and binding energy of TMAO-BSA conjugate using software AutoDock 4.2. Discovery studio, VMD and PyMol software have been used for visualization and identification of the amino acid residues in active site. Polar hydrogens including Kollmann charges were added, also Gasteiger charges were computed. The atomic type was assigned AD4 type. 2.4.3 Molecular Docking Finally, rigid molecular docking was performed with the optimized TMAO molecule using Autodock 4.2 tools. Genetic algorithm simulation program was used with a population size 300 and 100 runs. The remaining parameters were kept as it is to their default values. The output docked file was saved as Lamarckian GA. Top 10 confirmations of protein-ligand complex were saved on based on their negative binding energy (∆G) and RMSD values. PyMol and VMD and discovery studio softwares were taken into consideration to study the interaction and binding energy of EPS-BSA conjugate.[ 17 ] 2.4.3 MD Simulation and MMGBSA In our research to monitor the TMAO-BSA interaction, we have used Schorodinger suite for MD Simulation. Molecular dynamics production was performed till 150 ns with NPT (normal pressure & temperature) conditions. To know the real time interaction energy of TMAO-BSA complex we performed the post facto free-energy component analysis using MMGBSA. MM/GBSA approach is mainly used to compute the free energy and affinity of ligand binding with receptor molecule by using information related to structure through MD simulation.[ 18 ] OPLS_2005 has been used for force field parameters. Here the binding energy of the receptor and ligand as calculated by the Prime Energy i.e. a molecular mechanics and implicit solvent energy function (kcals/mol). 3. Results and Discussion Interaction between metabolite (TMAO) and BSA is studied using following spectroscopic techniques. 3.1 UV-Visible spectroscopy and PL study UV–visible absorbance spectra of TMAO, BSA and TMAO-BSA (deducting TMAO absorbance spectrum in acetate buffer, pH 4.3) solutions measured separately to ensure the mechanism of quenching. Figure_2 (a) shows that absorbance intensity of BSA around 280 nm decreases with the stepwise addition of TMAO solution in different concentrations. This suggests that quenching in absorbance intensity of BSA occurred mainly due to TMAO-BSA conjugate formation[ 19 ]. The weak peak obtained at 280 nm resulted from the aromatic amino acids (Try, Tyr and Phe) in protein structure. Proteins absorb UV radiation proportional to the amino acid content present in it. Therefore, it is possible to quantify proteins depending upon the absorption intensity. Aromatic amino acids like tryptophan and tyrosine shows absorbance of UV light at 280 nm, which is due to presence of aromatic ring structure in their side chain R group. Delocalization of π electrons takes place within the aromatic ring that is responsible for the absorption of light by aromatic amino acid residues. Benesi-Hildebrand plot (Figure_2 (b)) was derived from the UV-Visible plot (A/A 0 -A v/s 1/ [TMAO]) to calculate the total number of binding sites located on the BSA structure to associate with the metabolites. TMAO Slope of this plot gives the value of number of binding sites that is 4. The obtained values are in well agreement with the computational data. The phenomenon of quenching of fluorescence is decrement of quantum yield of fluorophore fluorescence induced by various types of interactions at molecular level, like ground-state conjugate formation, excited-state reactions, quenching due to collision and energy transfer[ 19 ]–[ 23 ]. By measuring intrinsic PL intensity of BSA protein before and after adding TMAO, there are some changes in microenvironment in proximity of fluorophore molecules. (Fig. 2 (c)) depicts the PL spectrum of BSA in the presence of different concentrations of TMAO at room temperature. When various amount TMAO was mixed with BSA solution which is fixed in concentration, the PL intensity of BSA around 280 nm observed to be decreasing in regular pattern but λ max (maximum emission intensity) remained unchanged did not change to either longer or shorter wavelength. Figure_2: (a) UV-Vis absorbance spectra recorded for various concentration of TMAO in fix concentration of BSA, (b) Benesi-Hildebrand plot UV-Visible plot (A/A 0 -A v/s 1/ [TMAO]) to calculate the total number of binding sites located on the BSA structure to associate with the TMAO. (c) PL spectra of BSA in the absence and presence of TMAO at different concentration (d) Stern–Volmer (SV) plot (e) Fo/F v/s [TMAO]. This signifies that TMAO might have interacted with BSA and quenched its intrinsic PL intensity, but here there was not any changes observed in local dielectric micro-environment of BSA. (Figure_2 (d & e)) shows to Stern–Volmer (SV) plot at room temperature. Graph shows that for the given investigated range of concentrations, the SV plot shows a good linearity. The Stern-Volmer constant (Ksv) may be obtained from both static as well as dynamic components. SV equation related to static quenching is formulated by taking consideration of conjugate formation between fluorophore molecule (F) and quencher molecule (Q) as reversible reaction along with association constant (K). But where there is dominancy of static quenching, the Ksv may be considered to be equal to association constant (K) between fluorophore and quencher molecule[ 24 ]–[ 26 ]. The intensity of fluorescence is considered as directly proportional to the concentration of fluorophore. The initial concentration of fluorophore [F o ], is equivalent to addition of the free fluorophore concentration [F], and no-fluorescent conjugate [FQ]. This gives linearity for Stern-Volmer equation for static quenching. \(\:\frac{F˳}{F}=1+Ksv\:\left[Q\right]\) ………………………………………………………………….…………….[ 1 ] where F 0 is the PL intensity in absence of quencher molecule, while F in the presence of quencher molecule. K SV is SV quenching constant and [Q] is the quencher concentration. Here results show an excellent linear correlation between (F 0 /F 1 ) and [TMAO]. The coefficient K SV is equal to the slope of this line; in this case, the value is 0.37. From the above discussion it clear that here static quenching of BSA has been occurred induced by TMAO. The data of PL quenching of BSA protein was analyzed to find the several binding factors. The total number of binding pocket (n) and binding constant (K b ) may be calculated according to below equation [ 27 ] $$\:Log\:\left[\frac{F˳-F}{F}\right]=\text{log}K\text{b}+n\text{log}\left[Q\right]$$ 2 …………………………………………………………… Here F o and F are fluorescent intensities in absence and presence of quencher molecule in steady state. From the Eq. ( 2 ), values of n and K b at room temperature were obtained to be 1.01 and 0.28 respectively. This implies that TMAO molecule is strongly bound to BSA. Also, here there is one independent class of binding pocket for TMAO molecule towards BSA. The linear- coefficient (R) is 0.96 which indicates that the underlying assumptions of derivation for Eq. ( 2 ) was satisfactory. To determine the interaction force between TMAO and BSA protein, the signs as well as magnitudes of the thermodynamic parameter (∆G) are accountable for the main interaction forces involved in binding process. The force of interaction between ligands and bio-macromolecules includes multiple hydrogen bonds, hydrophobic interaction, electrostatic interactions and van der Waals forces etc. Change in free energy (∆G) was further estimated from the equation mentioned below: $$\:\varDelta\:G=\:-RT\:ln\:Kb$$ where K b and R represents binding constant and gas constant (8.314 J/Mol/K) respectively. The negative value of ∆G indicates the spontaneity of the reaction between metabolite and BSA. The negative value of ∆G (-3.15 KJ) revealed that binding process is spontaneous. 3.3 CD study To observe the secondary structural change in BSA protein after interacting with ligand molecule, circular dichroism spectroscopy technique was used [ 28 ]. Figure 3 shows the CD spectrum of the BSA interaction with different concentration of the TMAO in acetate buffer pH = 4.3 at room temperature. Figure_3 clearly shows that BSA is exhibiting two negative peaks. One at 208 and other at 222 nm in ultraviolet zone, which is characteristics of typical α-helix structure of BSA protein. Both peaks 208 as well as 222 nm both contributing towards n→π* transition for the given peptide bonds of α-helices. As the concentration of TMAO is increased here, the intensity curves of given decreases in regular pattern from A–H (Fig. 3, band intensity curves A–H). The CD spectra of BSA protein were taken in the presence as well as in absence of TMAO that are showing similar shape of BSA indicating that BSA structure is still predominating the α-helices. Figure_3: CD spectra of BSA recorded in the presence and absence of TMAO at various concentration. 3.4 FTIR To confirm the interaction between TMAO-BSA conjugate, the FTIR spectral analysis was used. The compositions of pure BSA, TMAO dissolved in acetate buffer pH = 4.3, and TMAO-BSA conjugate were tested separately. Figure_4 shows the comparison of the FTIR spectra for pure BSA, TMAO in acetate buffer and TMAO-BSA conjugate [ 29 ], [ 30 ]. Figure_4: FTIR spectra of bovine serum albumin recorded in the presence and absence of TMAO at various concentration. Table 1 Transmission peaks in FTIR and their respective assignment in BSA, TMAO and TMAO-BSA conjugate. Wavenumber (cm − 1 ) Assigned To TMAO BSA TMAO + BSA 956 C–C bond out of the plane deformation √ √ √ 1075 C-OH stretch √ (due to acetate buffer) -- √ 1146 C-N bending √ √ Shifted to 1139 cm − 1 1243 N-O in N-Oxides √ Shifted to 1253 cm − 1 1344 N–H and C–H in-plane deformation √ √ √ 1400 C–N stretching vibration √ 1465 CH 3 deformation stretch -- √ -- 1562 N-O in aliphatic nitro compounds √ √ Shifted to 1548 cm − 1 1640 C = O stretch √ (due to acetate buffer) √ √ 1656 NH 2 stretch -- √ √ 1735 C = O stretch -- √ √ 1964 C = C Antisymmetric Stretch √ √ √ 2030 \(\:N-H\) deformation stretch √ √ √ 2150 N-C Stretch √ √ √ There are few peaks in the fingerprint region from 900 to 1650 cm − 1 which were present in all three spectra corresponding to C-OH, C-N and COO- groups that are due to both BSA and TMAO. The CH stretch in aliphatic compounds appeared between 2800–3000 cm − 1 which were present in all three spectra. The broad peaks appeared above 3000 cm − 1 correspond to OH and NH stretch vibrations. From the above table it is also observed that there is a shift in few peaks at 1146, 1243 and 1562 cm − 1 in conjugation spectrum indicating the interaction between both the components. Thus, FTIR spectrum confirmed the formation of conjugation between TMAO and BSA. 3.5 Contact Angle To know the hydrophobic or hydrophilic nature of TMAO-BSA complex the measurement of contact angle of BSA, TMAO and conjugates (BSA: TMAO) were carried out on ELISA plate. BSA protein is both hydrophobic as well as hydrophilic in nature. The hydrophilicity of BSA is mainly contributed due to lysine amino acid residue that contains positively charged amino group. Also arginine, glutamine, and asparagine have polar or charged side chains that may interact favorably with water molecules through hydrogen bonding and electrostatic interactions, making them hydrophilic. Amino acids with hydroxyl groups like serine and threonine and amino acids with amide groups such as asparagine and glutamine also contribute to the hydrophilic nature of BSA. The hydrophobic nature of BSA protein is primarily due to the presence of amino acids with nonpolar side chains, like alanine, valine, leucine, isoleucine, phenylalanine, and tryptophan. These amino acids have hydrophobic characteristics because their side chains lack charged or polar groups, making them unable to form favorable interactions with water molecules. Among these, amino acids like leucine, isoleucine, phenylalanine, and tryptophan are particularly hydrophobic due to the presence of bulky hydrocarbon side chains. These hydrophobic amino acids tend to cluster together in the interior of the protein's structure, away from the surrounding water molecules, contributing to the overall hydrophobicity of BSA. Here the contact angle formed by BSA molecules with ELISA plate is 91 ᵒ that means here it is involved in hydrophobic-hydrophobic interaction with the plate surface in acetate buffer solution. TMAO is amphiphilic molecule in nature as it has both hydrophilic and hydrophobic moieties [ 31 ]. Subsequently, Ɵ value for TMAO was also checked, which is less i.e. 82.95 o in comparison with BSA. This suggests the TMAO molecules form stronger bond with plate surface in comparison to the TMAO in acetate buffer. Thereafter, when TMAO was allowed to form conjugate with BSA molecules in different ratios from 1:1 to 1:100; the Ɵ value continuously decreased with the increasing ratio, respectively as shown in Fig. 5 (a to h). Figure_5 (a – h): Contact angle of bovine serum albumin and of TMAO-BSA conjugate at various concentration of TMAO. The hydrophilic nature of TMAO is due to its polar group which interacts with water molecule. When TMAO combines with BSA the resulting conjugate becomes more hydrophilic as observed from the above results. TMAO has been observed to stabilize proteins and maintain their solubility due to ability of forming hydrogen bonds with water molecules and protein surfaces. So the combination of TMAO with BSA is expected to enhance the hydrophilic nature of the TMAO-BSA conjugate. 3.6 Docking Results 3.6.1 Theoretical Modeling of TMAO and DFT Figure_6 (a) showing the ground state configuration of TMAO as the space-filling model after optimization as well as energy minimization. The energy value of optimized molecule was found to be E(RB3LYP) = -249.68522468 a.u., while RMS Gradient Norm = 0.00830651 a.u. with dipole moment = 4.6272 Debye. The greyish color balls represent C atoms, white color ball hydrogen atoms while ball with blue color symbolizes nitrogen atom and pink color represent oxygen atom of TMAO molecule. Figure_6: (a) 3D structure of ground state configuration of TMAO molecule, (b) Energy minimization and optimization graph of TMAO showing energy minimization with optimization step number (c) Energy minimization and optimization graph of TMAO showing energy RMS gradient normalization with optimization step number. The different bond lengths (in Å) and bond angles (°) for all possible confirmations of atoms present in TMAO molecules in 3D space has been mentioned in detail in table S1 and S2 respectively. 3.6.2 Docking of TMAO with BSA HIS 67 and GLU 243 were observed to be involved in conventional hydrogen bond formation with TMAO. Figure_7: shows the TMAO-BSA docked complex, having involvement of electrostatics and hydrogen bonding of TMAO with BSA Figure_8: (a) shows the lig plot structure of interaction of different amino acid residues with different atoms of TMAO. (c) 2D interactions and (d) 3D interaction of TMAO-BSA conjugate. GLU 243, ASP 248 and GLU 251 are involved in electrostatic interaction while LYS 242 is involved in carbon hydrogen bond as shown in Fig. 7. From docking result, it is clearly shown that the TMAO is binding with BSA. Since the TMAO is very small molecule so interaction is weak here as evident from the binding energy score value is -3.6 kcal/mol (shown in Table S3). 3.7.3 MD simulation and MMGBSA of TMAO with BSA The BSA protein and TMAO ligand RMSD value mentioned in figure S3 for 150 ns simulation. The ligand RMSD little fluctuating that means TMAO is found to be in the binding pocket of the protein but on the surface of the protein. In addition, the RMSD of BSA is not fluctuating so overall structure of protein remains relatively stable after binding with TMAO in TMAO-BSA complex. Figure_8: shows the involvement of amino acid residue of BSA in TMAO-BSA complex, having involvement of electrostatics and hydrogen, hydrophobic interaction and water bridges. A timeline representation of the interactions and contacts of TMAO with BSA that has been monitored throughout the simulation until 150 ns. The top panel shows the total number of specific contacts the protein makes with the ligand over the course of the trajectory. The bottom panel shows which residues interact with the ligand in each trajectory. Some residues make more than one specific contact with the ligand, which is represented by a darker shade of orange, according to the scale to the right of the plot. In our research, mainly four types of interaction like hydrogen bonds, hydrophobic, ionic and water bridges have been overserved in TMAO-BSA complex as shown in the plot above in the Figure_8. Each interaction type contains subtypes that are more specific. The stacked bar charts are normalized over the course of the trajectory. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the TMAO molecule. Consideration of hydrogen-bonding properties in TMAO- BSA interaction is important because of their strong influence on specificity, metabolization and adsorption. Hydrogen bonds between TMAO and BSA are backbone acceptor; backbone donor; side-chain acceptor; side-chain donor. Hydrophobic interactions are like π-Cation; π-π; and non-specific interactions. Ionic interactions are between two oppositely charged atoms that are within 3.7 Å of each other and do not involve a hydrogen bond. Water Bridges are hydrogen-bonded protein-ligand interactions mediated by a water molecule. A schematic of detailed interactions of TMAO atoms with the BSA amino acid residues, that occur more than 30.0% of the simulation time in the selected trajectory for 150.30 nsec), are shown. It is possible to interact with > 100% as some residues may have multiple interactions of a single type with the same ligand atom. The ∆G calculated through MMGBSA was found to be more negative that suggests that TMAO is binding with BSA forming TMAO-BSA complex. 4. Conclusion This manuscript describes about the interaction of TMAO with BSA with the help of various optical techniques and computational methods. Both the results obtained from experimental spectroscopic methods and in silico docking studies are in well agreement with each other. UV-Vis absorbance spectroscopy indicated the absorbance quenching of BSA with the increasing concentration of TMAO which could mainly be due to TMAO-BSA conjugate formation. The decrease in intensity of fluorescence is mainly due to involvement of tryptophan during the conjugation. The occurrence of static quenching was confirmed via Stern–Volmer (SV) plot, which consider the complex formation between fluorophore and protein molecule. The negative value for ∆G revealed that binding process is spontaneous in this case i.e., conjugate formation was occurred. From insilco analysis, i.e. molecular docking, MD simulation and MMGBSA analysis we concluded that four major types of interactions like hydrogen bonding, electrostatic interaction, hydrophobic interactions and water bridges are are the major factor for stabilizing the TMAO-BSA complex. The ∆G calculated through MMGBSA was found to be more negative that suggests that TMAO is binding with BSA forming TMAO-BSA complex. The link of TMAO with various diseases such as diabetes, renal and cardiovascular disease, thus aptamer against TMAO could be helpful in early diagnosis of the diseases related to it. Declarations Acknowledgments The authors acknowledge funding support from core grant received from the National Institute of Immunology, New Delhi. Authors would also acknowledge the SCFBio and School of Bioengineering and Biosciences LPU, for providing the computational facility. Contribution of Authors AKV has performed experiments, analyzed the data and wrote the manuscript. PG has helped in experimental work. AM and NR have edited the manuscript and provided the support for MD simulation. GBVS has helped in FTIR data analysis as well as reviewed the draft of manuscript. AK and PS conceptualized and designed the work and edited the manuscript to its final shape. Information of Author *Corresponding author: E-mail: [email protected] Conflict of Interest There is no any kind of conflict of interest among all authors Funding declaration Funding Institution – The fund will be granted through the National Institute of Immunology, New Delhi, India References Zeisel SH, Warrier M. Trimethylamine N-oxide, the microbiome, and heart and kidney disease. Annu Rev Nutr. 2017;37:157–81. Subramaniam S, Fletcher C. Trimethylamine N-oxide: breathe new life. Br J Pharmacol. 2018;175(8):1344–53. Ufnal M, Zadlo A, Ostaszewski R. TMAO: A small molecule of great expectations. Nutrition. 2015;31:11–2. 10.1016/j.nut.2015.05.006 . Yancey PH. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J Exp Biol. 2005;208(15):2819–30. Li X, Yang Z. Interaction of oridonin with human serum albumin by isothermal titration calorimetry and spectroscopic techniques. Chem Biol Interact. 2015;232:77–84. Bi S, et al. Investigation of the interaction between flavonoids and human serum albumin. J Mol Struct. 2004;703:1–3. Michnik A, Michalik K, Kluczewska A, Drzazga Z. Comparative DSC study of human and bovine serum albumin. J Therm Anal Calorim. 2006;84(1):113–7. Yue Y, Liu J, Fan J, Yao X. Binding studies of phloridzin with human serum albumin and its effect on the conformation of protein. J Pharm Biomed Anal. 2011;56(2):336–42. Seedher N, Agarwal P. Complexation of fluoroquinolone antibiotics with human serum albumin: A fluorescence quenching study. J Lumin. 2010;130(10):1841–8. Chaves OA, et al. Fluorescence and docking studies of the interaction between human serum albumin and pheophytin. Molecules. 2015;20(10):19526–39. Gulati P, kumar Verma A, Kumar A, Solanki P. Para-Cresyl Sulfate and BSA Conjugation for Developing Aptasensor: Spectroscopic Methods and Molecular Simulation. ECS J Solid State Sci Technol. 2023;12(7):73004. 10.1149/2162-8777/ace286 . Li S, Tonelli M, Unsworth LD. Indoxyl and p-cresol sulfate binding with human serum albumin. Colloids Surf Physicochem Eng Asp. 2022;635:128042. Sułkowska A. Interaction of drugs with bovine and human serum albumin. J Mol Struct. 2002;614:1–3. Zhang Y, Shi S, Chen X, Zhang W, Huang K, Peng M. Investigation on the interaction between ilaprazole and bovine serum albumin without or with different C-ring flavonoids from the viewpoint of food–drug interference. J Agric Food Chem. 2011;59(15):8499–506. Zhang YZ, Xiang X, Mei P, Dai J, Zhang LL, Liu Y. Spectroscopic studies on the interaction of Congo Red with bovine serum albumin. Spectrochim Acta - Part Mol Biomol Spectrosc. 2009;72(4):907–14. 10.1016/j.saa.2008.12.007 . Verma AK, Sharma S, Jayaraj A, Deep S. In silico study of interaction of (ZnO) 12 nanocluster to glucose oxidase-FAD in absence and presence of glucose. J Biomol Struct Dyn, pp. 1–9, 2023. Verma AK, Mishra A, Kumar T, Sardar M, Solanki PR. Experimental and In Silico interaction studies of Alpha Amylase-Silver nanoparticle: a nano-bio-conjugate, pp. 1–17, 2022. Genheden S. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinity, Accessed: Nov. 26, 2018. [Online]. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487606/pdf/iedc-10-449.pdf Wang YQ, Zhang HM, Zhang GC, Tao WH, Tang SH. Interaction of the flavonoid hesperidin with bovine serum albumin: A fluorescence quenching study. J Lumin. 2007;126(1):211–8. 10.1016/j.jlumin.2006.06.013 . Verma AK. ZnO quantum dots a novel nanomaterial for various applications: Recent advances and challenges. Indian J Biochem Biophys. 2022;59(12):1190–8. 10.56042/ijbb.v59i12.67314 . Verma AK. Application of Fluorescence Quantum Dots to Understand the Interaction between Nanomaterials and Biomolecules. Verma AK, Ansari ZA. Fluorescent ZnO Quantum Dot Probe to Study Glucose–Glucose Oxidase Interaction via Fluorescence Resonance Energy Transfer. Sens Lett. 2020;18(5):351–65. Verma AK, Dhiman TK, Lakshmi G, Solanki P. Optical tuning of polymer functionalized zinc oxide quantum dots as a selective probe for specific detection of antibiotics. bioRxiv, 2022. Edition T. Conjugated Polymers: Theory, Synthesis, Properties, and Characterization , 3rd ed. 2006. Acquavella MF, Evans ME, Farraher SW, Névoret CJ, Abelt CJ. Static and dynamic fluorescence quenching of a dicyanoanthracene-capped β-cyclodextrin. J Chem Soc Perkin Trans. 1995;2(2):385–8. 10.1039/p29950000385 . Lakowicz JR. Principles of Fluorescence Spectroscopy Third Edition, 2006. Accessed: Nov. 27, 2018. [Online]. Available: http://nathan.instras.com/MyDocsDB/doc-800.pdf Kang J, Liu Y, Xie MX, Li S, Jiang M, Wang YD. Interactions of human serum albumin with chlorogenic acid and ferulic acid. Biochim Biophys Acta - Gen Subj. 2004;1674(2):205–14. 10.1016/j.bbagen.2004.06.021 . Haq SK, Khan RH. Spectroscopic analysis of thermal denaturation of Cajanus cajan proteinase inhibitor at neutral and acidic pH by circular dichroism. Int J Biol Macromol. 2005;35:1–2. 10.1016/j.ijbiomac.2004.12.005 . Maruyama T, et al. FT-IR analysis of BSA fouled on ultrafiltration and microfiltration membranes. J Memb Sci. 2001;192:1–2. 10.1016/S0376-7388(01)00502-6 . Li Z, Qiang L, Zhong S, Wang H, Cui X. Synthesis and characterization of monodisperse magnetic Fe3O4at BSA core-shell nanoparticles. Colloids Surf Physicochem Eng Asp. 2013;436:1145–51. 10.1016/j.colsurfa.2013.08.044 . Macdonald RD, Khajehpour M. Effects of the osmolyte TMAO (Trimethylamine-N-oxide) on aqueous hydrophobic contact-pair interactions. Biophys Chem. 2013;184:101–7. 10.1016/j.bpc.2013.10.001 . Additional Declarations No competing interests reported. Supplementary Files TMAOSupplementrayfile.pdf Cite Share Download PDF Status: Published Journal Publication published 21 Jan, 2025 Read the published version in BMC Chemistry → Version 1 posted Editorial decision: Revision requested 16 Oct, 2024 Reviews received at journal 14 Oct, 2024 Reviews received at journal 12 Oct, 2024 Reviewers agreed at journal 04 Oct, 2024 Reviewers agreed at journal 04 Oct, 2024 Reviewers invited by journal 04 Oct, 2024 Editor invited by journal 03 Oct, 2024 Editor assigned by journal 01 Oct, 2024 Submission checks completed at journal 01 Oct, 2024 First submitted to journal 29 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5176166","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370416822,"identity":"2b55671f-bc99-47a1-b6ff-ad0b00218655","order_by":0,"name":"Awadhesh Kumar Verma","email":"","orcid":"","institution":"Lovely Professional University","correspondingAuthor":false,"prefix":"","firstName":"Awadhesh","middleName":"Kumar","lastName":"Verma","suffix":""},{"id":370416823,"identity":"e9630586-5069-452c-9c94-57608aeff23e","order_by":1,"name":"Payal Gulati","email":"","orcid":"","institution":"National Institute of Immunology","correspondingAuthor":false,"prefix":"","firstName":"Payal","middleName":"","lastName":"Gulati","suffix":""},{"id":370416824,"identity":"61aa8cfb-598b-4949-909b-d1220f235c33","order_by":2,"name":"GBVS Lakshmi","email":"","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":false,"prefix":"","firstName":"GBVS","middleName":"","lastName":"Lakshmi","suffix":""},{"id":370416825,"identity":"c9855895-4198-4c7a-bc06-d08bae341b11","order_by":3,"name":"Anand Mohan","email":"","orcid":"","institution":"Lovely Professional University","correspondingAuthor":false,"prefix":"","firstName":"Anand","middleName":"","lastName":"Mohan","suffix":""},{"id":370416827,"identity":"7831301c-5dc0-4f5c-b7bb-cfa3129f9363","order_by":4,"name":"Neeta Raj Sharma","email":"","orcid":"","institution":"Lovely Professional University","correspondingAuthor":false,"prefix":"","firstName":"Neeta","middleName":"Raj","lastName":"Sharma","suffix":""},{"id":370416828,"identity":"328898f6-7c13-47e2-8e61-e9d2774a9c04","order_by":5,"name":"Pratima R Solanki","email":"","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":false,"prefix":"","firstName":"Pratima","middleName":"R","lastName":"Solanki","suffix":""},{"id":370416829,"identity":"95f1bd10-8a6d-40ab-b76d-2be469640832","order_by":6,"name":"Anil Kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYFACxsaDDRUMPGxgjgFxWhoONpwhTQsDw8HGNlKcxT+7ueHgzHl3ZPgYmB9+YCi4Q1iLxJ2DDQc3bnsGdBibsQSDwTMirLmR2HDw4bbDIL+YAf1ymLAOebCWOSAt7N+I02IA0rKxAaSFh0hbDEFaZhwDamHmKZZIIEaL3I30hw97ag7by7e3b/zw4Q8RWhCAGYgTSNEwCkbBKBgFowA3AAD3ojpAYT6L4AAAAABJRU5ErkJggg==","orcid":"","institution":"National Institute of Immunology","correspondingAuthor":true,"prefix":"","firstName":"Anil","middleName":"","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2024-09-29 17:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5176166/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5176166/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13065-024-01375-0","type":"published","date":"2025-01-21T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68351430,"identity":"ae580276-9454-4f24-820a-4b7e98b066e0","added_by":"auto","created_at":"2024-11-06 10:44:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":740075,"visible":true,"origin":"","legend":"\u003cp\u003eFormation of TMAO from various precursor obtained from ingested foods and its implications in different diseases.\u003c/p\u003e","description":"","filename":"FIGURE1.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/6c0226d378f41a78baa16aa5.png"},{"id":68351431,"identity":"8e1f4be1-b3ad-4533-9dde-f9b86c3e53de","added_by":"auto","created_at":"2024-11-06 10:44:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":382000,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV-Vis absorbance spectra recorded for various concentration of TMAO in fix concentration of BSA, (b) Benesi-Hildebrand plot UV-Visible plot (A/A\u003csub\u003e0\u003c/sub\u003e-A v/s 1/ [TMAO]) to calculate the total number of binding sites located on the BSA structure to associate with the TMAO. (c) PL spectra of BSA in the absence and presence of TMAO at different concentration (d) Stern–Volmer (SV) plot (e) Fo/F v/s [TMAO].\u003c/p\u003e","description":"","filename":"FIGURE2.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/b96eaf3b41c33cdbc4d6d7aa.png"},{"id":68351432,"identity":"900e3376-4200-4507-a1bf-4d2051c3249d","added_by":"auto","created_at":"2024-11-06 10:44:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":343558,"visible":true,"origin":"","legend":"\u003cp\u003eCD spectra of BSA recorded in the presence and absence of TMAO at various concentration.\u003c/p\u003e","description":"","filename":"FIGURE3.png","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/9e5fc66191284015e4845412.png"},{"id":68351805,"identity":"ba6452b1-3a64-440c-89de-8e194c73b775","added_by":"auto","created_at":"2024-11-06 10:52:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183360,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of bovine serum albumin recorded in the presence and absence of TMAO at various concentration.\u003c/p\u003e","description":"","filename":"FIGURE4.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/1fb30cd41313a89f1a96a226.png"},{"id":68351804,"identity":"840b1e47-76b3-4e22-8688-455af6016e4b","added_by":"auto","created_at":"2024-11-06 10:52:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":978447,"visible":true,"origin":"","legend":"\u003cp\u003e(a – h): Contact angle of bovine serum albumin and of TMAO-BSA conjugate at various concentration of TMAO.\u003c/p\u003e","description":"","filename":"FIGURE5.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/b88f7a2e8376855b00538e39.png"},{"id":68351807,"identity":"9a7d45cd-5d03-4e6a-b3c7-37b92fd86255","added_by":"auto","created_at":"2024-11-06 10:52:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":683334,"visible":true,"origin":"","legend":"\u003cp\u003e(a) 3D structure of ground state configuration of TMAO molecule, (b) Energy minimization and optimization graph of TMAO showing energy minimization with optimization step number (c) Energy minimization and optimization graph of TMAO showing energy RMS gradient normalization with optimization step number. The different bond lengths (in Å) and bond angles (°) for all possible confirmations of atoms present in TMAO molecules in 3D space has been mentioned in detail in table S1 and S2 respectively.\u003c/p\u003e","description":"","filename":"FIGURE6.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/5d2dd8ba12a9b2a5e926f3f9.png"},{"id":68351434,"identity":"2fc1dedc-1e94-4b54-9f4f-f4453f281cc7","added_by":"auto","created_at":"2024-11-06 10:44:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1555146,"visible":true,"origin":"","legend":"\u003cp\u003eshows the TMAO-BSA docked complex, having involvement of electrostatics and hydrogen bonding of TMAO with BSA Figure_8: (a) shows the lig plot structure of interaction of different amino acid residues with different atoms of TMAO. (c) 2D interactions and (d) 3D interaction of TMAO-BSA conjugate.\u003c/p\u003e","description":"","filename":"FIGURE7.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/b55285413611d48970a429a6.png"},{"id":68353026,"identity":"d88bde7e-e255-4e92-8c2a-270694680475","added_by":"auto","created_at":"2024-11-06 11:00:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":856478,"visible":true,"origin":"","legend":"\u003cp\u003eshows the involvement of amino acid residue of BSA in TMAO-BSA complex, having involvement of electrostatics and hydrogen, hydrophobic interaction and water bridges.\u003c/p\u003e","description":"","filename":"FIGURE8.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/067e3882632845b06dd901a6.png"},{"id":74858590,"identity":"e4242d9d-ddb3-4662-91bb-90d15f02f01c","added_by":"auto","created_at":"2025-01-27 16:11:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7109739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/e5b10a78-d08b-4b58-b85a-5a741ab4f683.pdf"},{"id":68351438,"identity":"6aafd704-d88b-4484-bd1d-f51486427f62","added_by":"auto","created_at":"2024-11-06 10:44:10","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":531153,"visible":true,"origin":"","legend":"","description":"","filename":"TMAOSupplementrayfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5176166/v1/0e68ab17c754c76a717c10b1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Interaction studies unveil potential binding sites on bovine serum albumin for gut metabolite trimethylamine n-oxide (TMAO)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe human gut is the primary source of Trimethylamine-N-oxide (TMAO). There, the gut microbiota breaks down food components into TMA, which is taken into the bloodstream via the intestinal mucosa. Hepatic flavin monooxygenases in the liver subsequently convert TMA into TMAO.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e],[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] As per recent research reports, circulating TMAO is becoming known to be associated with an increasing variety of other chronic conditions, such as cancer, type 2 diabetes mellitus (T2DM), and renal disease. (Fig.\u0026nbsp;1) The biological effects of TMAO vary among several animals and tissue types. Water-stressed organisms and tissues use TMAO as an organic osmolyte to preserve cell volume. The capacity of TMAO to modify the structure and activity of a broad class of physiologically significant molecules is one of its roles. TMAO plays a significant stabilizing role in nucleic acid and the folded state of proteins. Studies on the thermodynamic effects of TMAO on proteins have demonstrated that TMAO inhibits denaturation of proteins and mitigates the effects of heat and pressure.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] The accumulation of TMAO by the kidneys of mammals is a response to the destabilizing effects of urea (as well as inorganic ions) on macromolecular structures, such as proteins and nucleic acids, and to the inhibition of urea on activities, including ligand binding.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure_1: Formation of TMAO from various precursor obtained from ingested foods and its implications in different diseases.\u003c/p\u003e \u003cp\u003eThe main extracellular protein of the circulatory system, serum albumin, makes up roughly 60% of all plasma proteins. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] It transports various medications, fatty acids, thyroid hormones, and steroids and gut microbiota derived metabolites such as TMAO, TMA, indoxyl sulfate etc. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Because it is more readily available and less expensive, bovine serum albumin (BSA) is frequently used in place of human serum albumin (HSA) in laboratory investigations. Because of their 88% and 76% closeness in amino acid sequences, BSA and HSA have comparable binding capacities.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] Numerous studies have been conducted to examine the binding between gut microbial metabolites and natural chemicals with serum albumin.[\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] Because serum albumin is a drug's transport carrier and has a major impact on its bioavailability, research on the relationship between albumin and TMAO is crucial. The capacity of these gut metabolites to bind determines how successful they are as pharmacological agents. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Although TMAO has been extensively documented for its actions and implication in human health and diseases, no scientific data about its interaction with serum albumin is available.\u003c/p\u003e \u003cp\u003eThe interaction of TMAO and proteins such as bovine serum albumin (BSA) can involve physical binding, changes in protein conformation, and potential functional effects. As the mechanism behind specific binding of TMAO with Bovine serum albumin (BSA) and the effect of TMAO on protein confirmation are not clear. Hence, in the present study, efforts have been made to study these aspects in detail. It is evident that conformational change in serum albumin is prompted by interaction with metabolites such as TMAO, drugs molecules and dyes having lower molecular weight, that significantly alter the albumin secondary and tertiary structure. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] Therefore, binding of gut metabolite TMAO with serum albumin is very important in the field of biomedical research. This study investigated the binding between bovine serum albumin with TMAO. The binding relationship was investigated using multi-spectroscopic (UV-vis absorption, CD, fluorescence, FT-IR) and contact angle methods. In addition, molecular docking studies were performed to better visualize the binding mode with BSA. Study parameters included TMAO binding energies with BSA, complex formation, and binding site involvement.\u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents and Buffer\u003c/h2\u003e \u003cp\u003eBovine Serum Albumin (BSA) in lyophilized form, Tetramethyl ethylene diamine, Sodium dodecyl sulfate (SDS), Acryl amide, N, N methyl acrylamide, Bromo phenol blue and Trimethylamine N-Oxide (TMAO) with high purity were procured from Sigma Aldrich. Dialysis membrane (Snake Skin\u003csup\u003e\u0026trade;\u003c/sup\u003e Dialyis Tubing, 10K MWtO,16mm) was procured from Thermo Scientific. Glacial acetic acid was procured from TM Media. Sodium acetate was procured from Merck. Tris base was procured from G Bioscience. Ammonium per sulfate and glycine was procured from HiMedia. Comassie brilliant blue dye methanol and ethanol were procured from SRL Ranbaxy. 0.2-micron filter was procured from MDI and 2ml syringe was procured form HMD. MilliQ (18.2 MΩ\u0026middot;cm @ 25\u0026deg;C) from the Millipore water purification system was used for preparation of all the solutions. Acetate buffer at pH 4.3 was used for TMAO-BSA conjugate preparation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Instrumentation\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 UV-Vis Spectroscopy\u003c/h2\u003e \u003cp\u003eThe UV\u0026ndash;vis absorption spectra of BSA were taken in the absence and presence of TMAO using UV-2600, UV-VIS Spectro-photometer SHIMADZU (Shimadzu Corporation in Japan), through 1.0 cm quartz cells keeping slit width 5 nm and 600 nm/minute scan speed. The spectra were recorded in wavelength range between 200\u0026ndash;600 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 PL Studies\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFluorescence was measured with the help of Fluorescence spectrophotometer (Cary Eclipse, Model G9800A, Agilent Technology,) with fixed excitation as well as emission slit width (5 nm) and 600 nm/minute scan speed. A quartz cuvette (3.5 ml) with path length 10 mm was used.\u003c/span\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 FTIR\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTo determine the functional groups changes, residing on the surfaces of bio-conjugates; FTIR spectrophotometer were used in ATR mode (PerkinElmer Spectrum 1). The spectra were obtained in between range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 wavenumber, with scan speed of 32. The contact angle measurement was done using drop shape analyzer (KRUSS, Germany) to check the hydrophobicity/hydrophilicity of the TMAO-BSA conjugate on ELISA plate.\u003c/span\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 CD Spectroscopy\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCD spectroscopic study was performed monitor the effect of TMAO on secondary and tertiary structure of BSA in acetate buffer solution. CD spectra were obtained in the UV region using JASCO-1500 CD spectropolarimeter associated with a Peltier temperature controller (PTC-517). Prior to the measurement, instrument was calibrated routinely with D-10 camphor-sulfonic acid under N\u003c/span\u003e \u003csub\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e \u003c/sub\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003epurging in lamp compartment, optics and sample chamber in a ratio of 1:3:1 to remove oxygen content and create the inert atmosphere. CD spectra i.e., ellipticity of the acetate buffer solution was recorded as a reference/background in the UV range from 190\u0026ndash;250 nm using a cell (path length 1 mm) at room temperature (25\u0026deg;C), 1 nm bandwidth. The average of three measurements were displayed as final spectra with mean time 1 nm/sec.\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Conjugate Preparation\u003c/h2\u003e \u003cp\u003eTMAO (in acetate buffer, PH\u0026thinsp;=\u0026thinsp;4.3) was conjugated with BSA (acetate buffer, pH\u0026thinsp;=\u0026thinsp;4.3) in different molar ratio\u0026rsquo;s including 1:1, 1:5, 1:10, 1:25, 1:50, 1;75, 1:100. A 3.5 mM of TMAO was mixed with 2 \u0026micro;M of BSA and allowed to react for 2 h at room temperature and for 72 h at 4\u0026deg;C. At the end of the reaction, dialysis was performed for 72 h with change of acetate buffer (pH\u0026thinsp;=\u0026thinsp;4.3) after every 12 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Computational Details\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Molecular Modeling and optimization of TMAO using DFT\u003c/h2\u003e \u003cp\u003eWe have theoretically modeled 3D structure of gut metabolite TMAO using Marvin sketch software. Every time 2D and 3D cleaning was performed and 3D structural confirmation of the molecule was checked by visualizing them in Marvin view. Further, we calculated each and every parameter like atomic coordinates (XYZ), bond length, and bond angle using ChemBioDraw Ultra software and optimized the structure up to transition state followed by energy minimization using MM2. The 3D structures were optimized to transition state, and the energy was minimized sequentially using further optimization followed by energy minimization using Chemdraw and Chem3DPro. The Final optimization was done via DFT approach with Gaussian 09 software with standard basis set: 6-311G and RB3LYP functional. This optimized structure was further utilized for docking to monitor the interactions between TMAO and BSA molecule. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Preparation of Receptor molecule\u003c/h2\u003e \u003cp\u003eBSA structure (Source organism: \u003cem\u003eBos Taurus\u003c/em\u003e, resolution: 2.70 \u0026Aring; and having 583 amino acid residues) was retrieved from Protein Data Bank having RCSB PDB ID: 3V03. The structure was cleaned of crystallographic water molecules and other crystalizing agents. Docking was performed to find the binding affinity and binding energy of TMAO-BSA conjugate using software AutoDock 4.2. Discovery studio, VMD and PyMol software have been used for visualization and identification of the amino acid residues in active site. Polar hydrogens including Kollmann charges were added, also Gasteiger charges were computed. The atomic type was assigned AD4 type.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Molecular Docking\u003c/h2\u003e \u003cp\u003eFinally, rigid molecular docking was performed with the optimized TMAO molecule using Autodock 4.2 tools. Genetic algorithm simulation program was used with a population size 300 and 100 runs. The remaining parameters were kept as it is to their default values. The output docked file was saved as Lamarckian GA. Top 10 confirmations of protein-ligand complex were saved on based on their negative binding energy (∆G) and RMSD values. PyMol and VMD and discovery studio softwares were taken into consideration to study the interaction and binding energy of EPS-BSA conjugate.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 MD Simulation and MMGBSA\u003c/h2\u003e \u003cp\u003eIn our research to monitor the TMAO-BSA interaction, we have used Schorodinger suite for MD Simulation. Molecular dynamics production was performed till 150 ns with NPT (normal pressure \u0026amp; temperature) conditions. To know the real time interaction energy of TMAO-BSA complex we performed the post facto free-energy component analysis using MMGBSA. MM/GBSA approach is mainly used to compute the free energy and affinity of ligand binding with receptor molecule by using information related to structure through MD simulation.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] OPLS_2005 has been used for force field parameters. Here the binding energy of the receptor and ligand as calculated by the Prime Energy i.e. a molecular mechanics and implicit solvent energy function (kcals/mol).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eInteraction between metabolite (TMAO) and BSA is studied using following spectroscopic techniques.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 UV-Visible spectroscopy and PL study\u003c/h2\u003e \u003cp\u003eUV\u0026ndash;visible absorbance spectra of TMAO, BSA and TMAO-BSA (deducting TMAO absorbance spectrum in acetate buffer, pH 4.3) solutions measured separately to ensure the mechanism of quenching. Figure_2 (a) shows that absorbance intensity of BSA around 280 nm decreases with the stepwise addition of TMAO solution in different concentrations. This suggests that quenching in absorbance intensity of BSA occurred mainly due to TMAO-BSA conjugate formation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The weak peak obtained at 280 nm resulted from the aromatic amino acids (Try, Tyr and Phe) in protein structure. Proteins absorb UV radiation proportional to the amino acid content present in it. Therefore, it is possible to quantify proteins depending upon the absorption intensity. Aromatic amino acids like tryptophan and tyrosine shows absorbance of UV light at 280 nm, which is due to presence of aromatic ring structure in their side chain R group. Delocalization of π electrons takes place within the aromatic ring that is responsible for the absorption of light by aromatic amino acid residues.\u003c/p\u003e \u003cp\u003eBenesi-Hildebrand plot (Figure_2 (b)) was derived from the UV-Visible plot (A/A\u003csub\u003e0\u003c/sub\u003e-A v/s 1/ [TMAO]) to calculate the total number of binding sites located on the BSA structure to associate with the metabolites. TMAO Slope of this plot gives the value of number of binding sites that is 4. The obtained values are in well agreement with the computational data.\u003c/p\u003e \u003cp\u003eThe phenomenon of quenching of fluorescence is decrement of quantum yield of fluorophore fluorescence induced by various types of interactions at molecular level, like ground-state conjugate formation, excited-state reactions, quenching due to collision and energy transfer[\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. By measuring intrinsic PL intensity of BSA protein before and after adding TMAO, there are some changes in microenvironment in proximity of fluorophore molecules. (Fig.\u0026nbsp;2 (c)) depicts the PL spectrum of BSA in the presence of different concentrations of TMAO at room temperature. When various amount TMAO was mixed with BSA solution which is fixed in concentration, the PL intensity of BSA around 280 nm observed to be decreasing in regular pattern but λ\u003csub\u003emax\u003c/sub\u003e (maximum emission intensity) remained unchanged did not change to either longer or shorter wavelength.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure_2: (a) UV-Vis absorbance spectra recorded for various concentration of TMAO in fix concentration of BSA, (b) Benesi-Hildebrand plot UV-Visible plot (A/A\u003csub\u003e0\u003c/sub\u003e-A v/s 1/ [TMAO]) to calculate the total number of binding sites located on the BSA structure to associate with the TMAO. (c) PL spectra of BSA in the absence and presence of TMAO at different concentration (d) Stern\u0026ndash;Volmer (SV) plot (e) Fo/F v/s [TMAO].\u003c/p\u003e \u003cp\u003eThis signifies that TMAO might have interacted with BSA and quenched its intrinsic PL intensity, but here there was not any changes observed in local dielectric micro-environment of BSA. (Figure_2 (d \u0026amp; e)) shows to Stern\u0026ndash;Volmer (SV) plot at room temperature. Graph shows that for the given investigated range of concentrations, the SV plot shows a good linearity. The Stern-Volmer constant (Ksv) may be obtained from both static as well as dynamic components. SV equation related to static quenching is formulated by taking consideration of conjugate formation between fluorophore molecule (F) and quencher molecule (Q) as reversible reaction along with association constant (K). But where there is dominancy of static quenching, the Ksv may be considered to be equal to association constant (K) between fluorophore and quencher molecule[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The intensity of fluorescence is considered as directly proportional to the concentration of fluorophore. The initial concentration of fluorophore [F\u003csub\u003eo\u003c/sub\u003e], is equivalent to addition of the free fluorophore concentration [F], and no-fluorescent conjugate [FQ]. This gives linearity for Stern-Volmer equation for static quenching.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{F˳}{F}=1+Ksv\\:\\left[Q\\right]\\)\u003c/span\u003e \u003c/span\u003e\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e \u003cp\u003ewhere F\u003csub\u003e0\u003c/sub\u003e is the PL intensity in absence of quencher molecule, while F in the presence of quencher molecule. K\u003csub\u003eSV\u003c/sub\u003e is SV quenching constant and [Q] is the quencher concentration. Here results show an excellent linear correlation between (F\u003csub\u003e0\u003c/sub\u003e/F\u003csub\u003e1\u003c/sub\u003e) and [TMAO]. The coefficient K\u003csub\u003eSV\u003c/sub\u003e is equal to the slope of this line; in this case, the value is 0.37.\u003c/p\u003e \u003cp\u003eFrom the above discussion it clear that here static quenching of BSA has been occurred induced by TMAO. The data of PL quenching of BSA protein was analyzed to find the several binding factors. The total number of binding pocket (n) and binding constant (K\u003csub\u003eb\u003c/sub\u003e) may be calculated according to below equation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:Log\\:\\left[\\frac{F˳-F}{F}\\right]=\\text{log}K\\text{b}+n\\text{log}\\left[Q\\right]$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u003c/p\u003e \u003cp\u003eHere F\u003csub\u003eo\u003c/sub\u003e and F are fluorescent intensities in absence and presence of quencher molecule in steady state. From the Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e2\u003c/span\u003e), values of n and K\u003csub\u003eb\u003c/sub\u003e at room temperature were obtained to be 1.01 and 0.28 respectively. This implies that TMAO molecule is strongly bound to BSA. Also, here there is one independent class of binding pocket for TMAO molecule towards BSA. The linear- coefficient (R) is 0.96 which indicates that the underlying assumptions of derivation for Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was satisfactory.\u003c/p\u003e \u003cp\u003eTo determine the interaction force between TMAO and BSA protein, the signs as well as magnitudes of the thermodynamic parameter (∆G) are accountable for the main interaction forces involved in binding process. The force of interaction between ligands and bio-macromolecules includes multiple hydrogen bonds, hydrophobic interaction, electrostatic interactions and van der Waals forces etc. Change in free energy (∆G) was further estimated from the equation mentioned below:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\varDelta\\:G=\\:-RT\\:ln\\:Kb$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere K\u003csub\u003eb\u003c/sub\u003e and R represents binding constant and gas constant (8.314 J/Mol/K) respectively. The negative value of ∆G indicates the spontaneity of the reaction between metabolite and BSA. The negative value of ∆G (-3.15 KJ) revealed that binding process is spontaneous.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 CD study\u003c/h2\u003e \u003cp\u003eTo observe the secondary structural change in BSA protein after interacting with ligand molecule, circular dichroism spectroscopy technique was used [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Figure\u0026nbsp;3 shows the CD spectrum of the BSA interaction with different concentration of the TMAO in acetate buffer pH\u0026thinsp;=\u0026thinsp;4.3 at room temperature. Figure_3 clearly shows that BSA is exhibiting two negative peaks. One at 208 and other at 222 nm in ultraviolet zone, which is characteristics of typical α-helix structure of BSA protein. Both peaks 208 as well as 222 nm both contributing towards n\u0026rarr;π* transition for the given peptide bonds of α-helices. As the concentration of TMAO is increased here, the intensity curves of given decreases in regular pattern from A\u0026ndash;H (Fig.\u0026nbsp;3, band intensity curves A\u0026ndash;H). The CD spectra of BSA protein were taken in the presence as well as in absence of TMAO that are showing similar shape of BSA indicating that BSA structure is still predominating the α-helices.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure_3: CD spectra of BSA recorded in the presence and absence of TMAO at various concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 FTIR\u003c/h2\u003e \u003cp\u003eTo confirm the interaction between TMAO-BSA conjugate, the FTIR spectral analysis was used. The compositions of pure BSA, TMAO dissolved in acetate buffer pH\u0026thinsp;=\u0026thinsp;4.3, and TMAO-BSA conjugate were tested separately. Figure_4 shows the comparison of the FTIR spectra for pure BSA, TMAO in acetate buffer and TMAO-BSA conjugate [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure_4: FTIR spectra of bovine serum albumin recorded in the presence and absence of TMAO at various concentration.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTransmission peaks in FTIR and their respective assignment in BSA, TMAO and TMAO-BSA conjugate.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWavenumber (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAssigned To\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTMAO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBSA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTMAO\u0026thinsp;+\u0026thinsp;BSA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;C bond out of the plane deformation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC-OH stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic; (due to acetate buffer)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC-N bending\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShifted to 1139 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN-O in N-Oxides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShifted to 1253 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1344\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026ndash;H and C\u0026ndash;H in-plane deformation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026ndash;N stretching vibration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e deformation stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN-O in aliphatic nitro compounds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShifted to 1548 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;O stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic; (due to acetate buffer)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1656\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNH\u003csub\u003e2\u003c/sub\u003e stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;O stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e--\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u0026thinsp;=\u0026thinsp;C Antisymmetric Stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:N-H\\)\u003c/span\u003e\u003c/span\u003e deformation stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN-C Stretch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026radic;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThere are few peaks in the fingerprint region from 900 to 1650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which were present in all three spectra corresponding to C-OH, C-N and COO- groups that are due to both BSA and TMAO. The CH stretch in aliphatic compounds appeared between 2800\u0026ndash;3000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which were present in all three spectra. The broad peaks appeared above 3000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to OH and NH stretch vibrations. From the above table it is also observed that there is a shift in few peaks at 1146, 1243 and 1562 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in conjugation spectrum indicating the interaction between both the components. Thus, FTIR spectrum confirmed the formation of conjugation between TMAO and BSA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Contact Angle\u003c/h2\u003e \u003cp\u003eTo know the hydrophobic or hydrophilic nature of TMAO-BSA complex the measurement of contact angle of BSA, TMAO and conjugates (BSA: TMAO) were carried out on ELISA plate. BSA protein is both hydrophobic as well as hydrophilic in nature. The hydrophilicity of BSA is mainly contributed due to lysine amino acid residue that contains positively charged amino group. Also arginine, glutamine, and asparagine have polar or charged side chains that may interact favorably with water molecules through hydrogen bonding and electrostatic interactions, making them hydrophilic. Amino acids with hydroxyl groups like serine and threonine and amino acids with amide groups such as asparagine and glutamine also contribute to the hydrophilic nature of BSA. The hydrophobic nature of BSA protein is primarily due to the presence of amino acids with nonpolar side chains, like alanine, valine, leucine, isoleucine, phenylalanine, and tryptophan. These amino acids have hydrophobic characteristics because their side chains lack charged or polar groups, making them unable to form favorable interactions with water molecules. Among these, amino acids like leucine, isoleucine, phenylalanine, and tryptophan are particularly hydrophobic due to the presence of bulky hydrocarbon side chains. These hydrophobic amino acids tend to cluster together in the interior of the protein's structure, away from the surrounding water molecules, contributing to the overall hydrophobicity of BSA. Here the contact angle formed by BSA molecules with ELISA plate is 91\u003csup\u003eᵒ\u003c/sup\u003e that means here it is involved in hydrophobic-hydrophobic interaction with the plate surface in acetate buffer solution. TMAO is amphiphilic molecule in nature as it has both hydrophilic and hydrophobic moieties [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Subsequently, Ɵ value for TMAO was also checked, which is less i.e. 82.95\u003csup\u003eo\u003c/sup\u003e in comparison with BSA. This suggests the TMAO molecules form stronger bond with plate surface in comparison to the TMAO in acetate buffer. Thereafter, when TMAO was allowed to form conjugate with BSA molecules in different ratios from 1:1 to 1:100; the Ɵ value continuously decreased with the increasing ratio, respectively as shown in Fig.\u0026nbsp;5 (a to h).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure_5 (a \u0026ndash; h): Contact angle of bovine serum albumin and of TMAO-BSA conjugate at various concentration of TMAO.\u003c/p\u003e \u003cp\u003eThe hydrophilic nature of TMAO is due to its polar group which interacts with water molecule.\u003c/p\u003e \u003cp\u003eWhen TMAO combines with BSA the resulting conjugate becomes more hydrophilic as observed from the above results. TMAO has been observed to stabilize proteins and maintain their solubility due to ability of forming hydrogen bonds with water molecules and protein surfaces. So the combination of TMAO with BSA is expected to enhance the hydrophilic nature of the TMAO-BSA conjugate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Docking Results\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1 Theoretical Modeling of TMAO and DFT\u003c/h2\u003e \u003cp\u003eFigure_6 (a) showing the ground state configuration of TMAO as the space-filling model after optimization as well as energy minimization. The energy value of optimized molecule was found to be E(RB3LYP) = -249.68522468 a.u., while RMS Gradient Norm\u0026thinsp;=\u0026thinsp;0.00830651 a.u. with dipole moment\u0026thinsp;=\u0026thinsp;4.6272 Debye. The greyish color balls represent C atoms, white color ball hydrogen atoms while ball with blue color symbolizes nitrogen atom and pink color represent oxygen atom of TMAO molecule.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure_6: (a) 3D structure of ground state configuration of TMAO molecule, (b) Energy minimization and optimization graph of TMAO showing energy minimization with optimization step number (c) Energy minimization and optimization graph of TMAO showing energy RMS gradient normalization with optimization step number. The different bond lengths (in \u0026Aring;) and bond angles (\u0026deg;) for all possible confirmations of atoms present in TMAO molecules in 3D space has been mentioned in detail in table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2 respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2 Docking of TMAO with BSA\u003c/h2\u003e \u003cp\u003eHIS 67 and GLU 243 were observed to be involved in conventional hydrogen bond formation with TMAO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure_7: shows the TMAO-BSA docked complex, having involvement of electrostatics and hydrogen bonding of TMAO with BSA Figure_8: (a) shows the lig plot structure of interaction of different amino acid residues with different atoms of TMAO. (c) 2D interactions and (d) 3D interaction of TMAO-BSA conjugate.\u003c/p\u003e \u003cp\u003eGLU 243, ASP 248 and GLU 251 are involved in electrostatic interaction while LYS 242 is involved in carbon hydrogen bond as shown in Fig.\u0026nbsp;7. From docking result, it is clearly shown that the TMAO is binding with BSA. Since the TMAO is very small molecule so interaction is weak here as evident from the binding energy score value is -3.6 kcal/mol (shown in Table S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.7.3 MD simulation and MMGBSA of TMAO with BSA\u003c/h2\u003e \u003cp\u003eThe BSA protein and TMAO ligand RMSD value mentioned in figure S3 for 150 ns simulation. The ligand RMSD little fluctuating that means TMAO is found to be in the binding pocket of the protein but on the surface of the protein. In addition, the RMSD of BSA is not fluctuating so overall structure of protein remains relatively stable after binding with TMAO in TMAO-BSA complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure_8: shows the involvement of amino acid residue of BSA in TMAO-BSA complex, having involvement of electrostatics and hydrogen, hydrophobic interaction and water bridges.\u003c/p\u003e \u003cp\u003eA timeline representation of the interactions and contacts of TMAO with BSA that has been monitored throughout the simulation until 150 ns. The top panel shows the total number of specific contacts the protein makes with the ligand over the course of the trajectory. The bottom panel shows which residues interact with the ligand in each trajectory. Some residues make more than one specific contact with the ligand, which is represented by a darker shade of orange, according to the scale to the right of the plot. In our research, mainly four types of interaction like hydrogen bonds, hydrophobic, ionic and water bridges have been overserved in TMAO-BSA complex as shown in the plot above in the Figure_8. Each interaction type contains subtypes that are more specific. The stacked bar charts are normalized over the course of the trajectory. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the TMAO molecule. Consideration of hydrogen-bonding properties in TMAO- BSA interaction is important because of their strong influence on specificity, metabolization and adsorption. Hydrogen bonds between TMAO and BSA are backbone acceptor; backbone donor; side-chain acceptor; side-chain donor. Hydrophobic interactions are like π-Cation; π-π; and non-specific interactions. Ionic interactions are between two oppositely charged atoms that are within 3.7 \u0026Aring; of each other and do not involve a hydrogen bond. Water Bridges are hydrogen-bonded protein-ligand interactions mediated by a water molecule. A schematic of detailed interactions of TMAO atoms with the BSA amino acid residues, that occur more than 30.0% of the simulation time in the selected trajectory for 150.30 nsec), are shown. It is possible to interact with \u0026gt;\u0026thinsp;100% as some residues may have multiple interactions of a single type with the same ligand atom. The ∆G calculated through MMGBSA was found to be more negative that suggests that TMAO is binding with BSA forming TMAO-BSA complex.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":" \u003cp\u003eThis manuscript describes about the interaction of TMAO with BSA with the help of various optical techniques and computational methods. Both the results obtained from experimental spectroscopic methods and \u003cem\u003ein silico\u003c/em\u003e docking studies are in well agreement with each other. UV-Vis absorbance spectroscopy indicated the absorbance quenching of BSA with the increasing concentration of TMAO which could mainly be due to TMAO-BSA conjugate formation. The decrease in intensity of fluorescence is mainly due to involvement of tryptophan during the conjugation. The occurrence of static quenching was confirmed via Stern\u0026ndash;Volmer (SV) plot, which consider the complex formation between fluorophore and protein molecule. The negative value for ∆G revealed that binding process is spontaneous in this case i.e., conjugate formation was occurred. From \u003cem\u003einsilco\u003c/em\u003e analysis, i.e. molecular docking, MD simulation and MMGBSA analysis we concluded that four major types of interactions like hydrogen bonding, electrostatic interaction, hydrophobic interactions and water bridges are are the major factor for stabilizing the TMAO-BSA complex. The ∆G calculated through MMGBSA was found to be more negative that suggests that TMAO is binding with BSA forming TMAO-BSA complex. The link of TMAO with various diseases such as diabetes, renal and cardiovascular disease, thus aptamer against TMAO could be helpful in early diagnosis of the diseases related to it.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge funding support from core grant received from the National Institute of Immunology, New Delhi. Authors would also acknowledge the SCFBio and School of Bioengineering and Biosciences LPU, for providing the computational facility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution of Authors\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAKV has performed experiments, analyzed the data and wrote the manuscript. PG has helped in experimental work. AM and NR have edited the manuscript and provided the support for MD simulation. GBVS has helped in FTIR data analysis as well as reviewed the draft of manuscript. AK and PS conceptualized and designed the work and edited the manuscript to its final shape.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformation of Author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e*Corresponding author: E-mail:\u0026nbsp;[email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no any kind of conflict of interest among all authors\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding Institution \u0026ndash; The fund will be granted through the National Institute of Immunology, New Delhi, India\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZeisel SH, Warrier M. Trimethylamine N-oxide, the microbiome, and heart and kidney disease. Annu Rev Nutr. 2017;37:157\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubramaniam S, Fletcher C. Trimethylamine N-oxide: breathe new life. Br J Pharmacol. 2018;175(8):1344\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUfnal M, Zadlo A, Ostaszewski R. TMAO: A small molecule of great expectations. Nutrition. 2015;31:11\u0026ndash;2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.nut.2015.05.006\u003c/span\u003e\u003cspan address=\"10.1016/j.nut.2015.05.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYancey PH. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J Exp Biol. 2005;208(15):2819\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Yang Z. Interaction of oridonin with human serum albumin by isothermal titration calorimetry and spectroscopic techniques. Chem Biol Interact. 2015;232:77\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBi S, et al. Investigation of the interaction between flavonoids and human serum albumin. J Mol Struct. 2004;703:1\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichnik A, Michalik K, Kluczewska A, Drzazga Z. Comparative DSC study of human and bovine serum albumin. J Therm Anal Calorim. 2006;84(1):113\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYue Y, Liu J, Fan J, Yao X. Binding studies of phloridzin with human serum albumin and its effect on the conformation of protein. J Pharm Biomed Anal. 2011;56(2):336\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeedher N, Agarwal P. Complexation of fluoroquinolone antibiotics with human serum albumin: A fluorescence quenching study. J Lumin. 2010;130(10):1841\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaves OA, et al. Fluorescence and docking studies of the interaction between human serum albumin and pheophytin. Molecules. 2015;20(10):19526\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGulati P, kumar Verma A, Kumar A, Solanki P. Para-Cresyl Sulfate and BSA Conjugation for Developing Aptasensor: Spectroscopic Methods and Molecular Simulation. ECS J Solid State Sci Technol. 2023;12(7):73004. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1149/2162-8777/ace286\u003c/span\u003e\u003cspan address=\"10.1149/2162-8777/ace286\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Tonelli M, Unsworth LD. Indoxyl and p-cresol sulfate binding with human serum albumin. Colloids Surf Physicochem Eng Asp. 2022;635:128042.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSułkowska A. Interaction of drugs with bovine and human serum albumin. J Mol Struct. 2002;614:1\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Shi S, Chen X, Zhang W, Huang K, Peng M. Investigation on the interaction between ilaprazole and bovine serum albumin without or with different C-ring flavonoids from the viewpoint of food\u0026ndash;drug interference. J Agric Food Chem. 2011;59(15):8499\u0026ndash;506.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang YZ, Xiang X, Mei P, Dai J, Zhang LL, Liu Y. Spectroscopic studies on the interaction of Congo Red with bovine serum albumin. Spectrochim Acta - Part Mol Biomol Spectrosc. 2009;72(4):907\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.saa.2008.12.007\u003c/span\u003e\u003cspan address=\"10.1016/j.saa.2008.12.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma AK, Sharma S, Jayaraj A, Deep S. In silico study of interaction of (ZnO) 12 nanocluster to glucose oxidase-FAD in absence and presence of glucose. J Biomol Struct Dyn, pp. 1\u0026ndash;9, 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma AK, Mishra A, Kumar T, Sardar M, Solanki PR. Experimental and In Silico interaction studies of Alpha Amylase-Silver nanoparticle: a nano-bio-conjugate, pp. 1\u0026ndash;17, 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGenheden S. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinity, Accessed: Nov. 26, 2018. [Online]. Available: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487606/pdf/iedc-10-449.pdf\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487606/pdf/iedc-10-449.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang YQ, Zhang HM, Zhang GC, Tao WH, Tang SH. Interaction of the flavonoid hesperidin with bovine serum albumin: A fluorescence quenching study. J Lumin. 2007;126(1):211\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jlumin.2006.06.013\u003c/span\u003e\u003cspan address=\"10.1016/j.jlumin.2006.06.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma AK. ZnO quantum dots a novel nanomaterial for various applications: Recent advances and challenges. Indian J Biochem Biophys. 2022;59(12):1190\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.56042/ijbb.v59i12.67314\u003c/span\u003e\u003cspan address=\"10.56042/ijbb.v59i12.67314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma AK. Application of Fluorescence Quantum Dots to Understand the Interaction between Nanomaterials and Biomolecules.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma AK, Ansari ZA. Fluorescent ZnO Quantum Dot Probe to Study Glucose\u0026ndash;Glucose Oxidase Interaction via Fluorescence Resonance Energy Transfer. Sens Lett. 2020;18(5):351\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma AK, Dhiman TK, Lakshmi G, Solanki P. Optical tuning of polymer functionalized zinc oxide quantum dots as a selective probe for specific detection of antibiotics. bioRxiv, 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdition T. \u003cem\u003eConjugated Polymers: Theory, Synthesis, Properties, and Characterization\u003c/em\u003e, 3rd ed. 2006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAcquavella MF, Evans ME, Farraher SW, N\u0026eacute;voret CJ, Abelt CJ. Static and dynamic fluorescence quenching of a dicyanoanthracene-capped β-cyclodextrin. J Chem Soc Perkin Trans. 1995;2(2):385\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/p29950000385\u003c/span\u003e\u003cspan address=\"10.1039/p29950000385\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLakowicz JR. Principles of Fluorescence Spectroscopy Third Edition, 2006. Accessed: Nov. 27, 2018. [Online]. Available: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://nathan.instras.com/MyDocsDB/doc-800.pdf\u003c/span\u003e\u003cspan address=\"http://nathan.instras.com/MyDocsDB/doc-800.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang J, Liu Y, Xie MX, Li S, Jiang M, Wang YD. Interactions of human serum albumin with chlorogenic acid and ferulic acid. Biochim Biophys Acta - Gen Subj. 2004;1674(2):205\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbagen.2004.06.021\u003c/span\u003e\u003cspan address=\"10.1016/j.bbagen.2004.06.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaq SK, Khan RH. Spectroscopic analysis of thermal denaturation of Cajanus cajan proteinase inhibitor at neutral and acidic pH by circular dichroism. Int J Biol Macromol. 2005;35:1\u0026ndash;2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijbiomac.2004.12.005\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2004.12.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaruyama T, et al. FT-IR analysis of BSA fouled on ultrafiltration and microfiltration membranes. J Memb Sci. 2001;192:1\u0026ndash;2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0376-7388(01)00502-6\u003c/span\u003e\u003cspan address=\"10.1016/S0376-7388(01)00502-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Qiang L, Zhong S, Wang H, Cui X. Synthesis and characterization of monodisperse magnetic Fe3O4at BSA core-shell nanoparticles. Colloids Surf Physicochem Eng Asp. 2013;436:1145\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.colsurfa.2013.08.044\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfa.2013.08.044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMacdonald RD, Khajehpour M. Effects of the osmolyte TMAO (Trimethylamine-N-oxide) on aqueous hydrophobic contact-pair interactions. Biophys Chem. 2013;184:101\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bpc.2013.10.001\u003c/span\u003e\u003cspan address=\"10.1016/j.bpc.2013.10.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TMAO, bovine serum albumin, in silico, DFT, docking, spectroscopy, Molecular Dynamics simulation, mmgbsa","lastPublishedDoi":"10.21203/rs.3.rs-5176166/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5176166/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTrimethylamine-N-oxide (TMAO) is the most studied gut microbiota-derived metabolite and its binding to serum albumin has huge significance to understand the underlying mechanisms in human health and diseases. The interaction between BSA and TMAO has not been well studied; the binding mechanism, preferred binding locations, and subsequent conformation of BSA are still unclear. In the present work, spectroscopic, photoluminescence, Fourier transform infrared contact angle, circular dichroism, density functional theory, molecular docking, and molecular dynamics simulation approaches were used to probe TMAO-BSA interaction and identifying putative binding sites for TMAO on BSA. Non-covalent interaction of TMAO with bovine serum albumin forming stable docking complex with binding score of -3.6 kcal/mol was revealed through docking and simulation study. The docking complex of TMAO-BSA was found stable through hydrogen bond and electrostatic, Vander waals interaction, and water bridges via amino acid residues. Hydrophilicity/Hydrophobicity of the TMAO-BSA was studied using contact angle (Ɵ) measurement. Present study may be helpful for making strategies for Chronic Kidney Disease and other disease treatments wherein interaction of TMAO with serum albumin matters. Also, pharmacodynamics and pharmacokinetics of TMAO, as well as its mechanism of binding to BSA, may be better understood with the help of this study's findings.\u003c/p\u003e","manuscriptTitle":"Interaction studies unveil potential binding sites on bovine serum albumin for gut metabolite trimethylamine n-oxide (TMAO)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-06 10:44:05","doi":"10.21203/rs.3.rs-5176166/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-16T16:05:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-14T12:41:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-12T06:12:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329581494102287422735500104908632421004","date":"2024-10-04T05:35:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222308204131463072745505386571052414458","date":"2024-10-04T04:57:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-04T04:50:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-10-03T15:25:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-01T13:21:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-01T09:18:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Chemistry","date":"2024-09-29T17:36:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc2b2794-79cf-4fa2-92c4-8d8a0395c451","owner":[],"postedDate":"November 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-27T16:05:29+00:00","versionOfRecord":{"articleIdentity":"rs-5176166","link":"https://doi.org/10.1186/s13065-024-01375-0","journal":{"identity":"bmc-chemistry","isVorOnly":false,"title":"BMC Chemistry"},"publishedOn":"2025-01-21 15:57:15","publishedOnDateReadable":"January 21st, 2025"},"versionCreatedAt":"2024-11-06 10:44:05","video":"","vorDoi":"10.1186/s13065-024-01375-0","vorDoiUrl":"https://doi.org/10.1186/s13065-024-01375-0","workflowStages":[]},"version":"v1","identity":"rs-5176166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5176166","identity":"rs-5176166","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}