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Coherent Experimental and Simulation Approach To Explore the Underlying Mechanism of Denaturation of Stem Bromelain in Osmolytes Anjeeta Rani,† Mohamed Taha,‡ Pannuru Venkatesu,*,† and Ming- Jer Lee§ †

Department of Chemistry, University of Delhi, Delhi 110 007, India Department of Chemistry, College of Science, Sultan Qaboos University, PO Box 36, PC 123 Muscat, Oman § Department of Chemical Engineering, National Taiwan University of Science & Technology, Taipei 10607, Taiwan ‡

ABSTRACT: Characterization of a protein in the context of its environment is of crucial importance for a complete understanding of its function. Although biophysical techniques provide powerful tools for studying the stability and activity of the enzyme in the presence of various cosolvents, an approach of combining both experimental techniques and molecular dynamic (MD) simulations may lead to the mechanistic insight into the interactions governing the stability of an enzyme. The knowledge of these interactions can be further utilized for range of modifications in the wild form of an enzyme for various pharmaceutical applications. Herein, we employed florescence, UV−visible, circular dichroism (CD), dynamic light scattering (DLS) study, and MD simulations for comprehensive understanding of stem bromelain (BM) in the presence of betaine, sarcosine, arginine, and proline. The thermal stability of BM in the presence of 1 M of osmolytes is found to be in order: proline > betaine > buffer > arginine > sarcosine. BM gets more preferentially hydrated in the presence of betaine and proline than in sarcosine and arginine. Nonetheless, MD simulations suggest that betaine, sarcosine, and arginine at 1 M interact with the active site of BM through Hbonding except proline which are responsible for more disruption of active site. The distances between the catalytic site residues are 1.6, 1.9, 4.3, 5.0, and 6.2 Å for BM in proline, buffer, betaine, arginine, and sarcosine at 1 M, respectively. To the best of our knowledge, this is the first report on detailed unequivocal evidence of denaturation and deactivation of BM in the presence of methylamines and amino acids.

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denatured proteins.18,19 TMAO and betaine are the most ubiquitous osmolytes, present in large concentrations in intracellular fluids of many species of all kingdoms, however, betaine is also found in kidneys of several mammalian species where no TMAO is used to cope with the stresses.2,4 Amino acids are generally classified as compatible osmolytes. 2,4 Most of marine invertebrates, numerous prokaryotes and many mammalian cells utilize amino acids in order to survive in various harsh conditions.2,4,20,21 According to reviews by Hayat et al. and Szabados and Savouré,22,23 proline not only acts as an excellent osmolyte by maintaining osmotic balance and stabilizing membranes, but

INTRODUCTION By the accumulation of low molecular weight organic molecules, a diverse range of organisms from numerous phyla responded to the changes in intracellular water activity resulting from the hostile stresses such as dehydration, temperature variations, variable pH, freezing, high salinity, high concentrations of denaturants.1−6 These molecules are termed as osmolytes or osmoprotectants. The methylamines and amino acids are major classes of naturally occurring osmolytes. These are either nonperturbing or in some cases, favorable effects on the protein’s structural stability and functionality.1,2,4 The methylamines such as trimethylamine-Noxide (TMAO), betaine, and sarcosine have been shown to increase the thermodynamic stability of the proteins,7−11 to counteract the destabilizing effects of the denaturants, salts and hydrostatic pressure12−17 and to refold the partially © XXXX American Chemical Society

Received: February 23, 2017 Revised: June 12, 2017 Published: June 15, 2017 A

DOI: 10.1021/acs.jpcb.7b01776 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B

ism (CD), dynamic light scattering (DLS), and molecular dynamic (MD) simulations. To the best of our knowledge, this is the first report on an implication of a combination of experimental and simulation to the stability and activity of BM in the presence of betaine, sarcosine, proline, and arginine. Therefore, adoption of this study may help to resolve these uncovered aspects in literature which cannot be obviated from the necessity of today’s research.

also plays other roles during stress, i.e., metal chelator, antioxidative defense, signaling, stabilization of redox balance, and maintenance of cellular homeostasis, etc. Arginine is known to be exceptionally effective in curbing protein aggregations and can be appropriate for mounting the shelf life of proteins as a universal reagent.24 The literature reveals many interesting insights into the protein stabilization by the methylamines and amino acids.5,8−10,20,21,25,26 However, some of amino acids are found to be noncompatible with respect to protein stability, for example, arginine, lysine, and histidine, etc.21 Moreover, in the past decade, a number of research groups as well as our research group reported methylamines, which are well-known protein stabilizers, also as destabilizers for various proteins at different conditions.27,28 Despite the available literature, the reason behind these uncharacteristic behaviors is still not clear. In fact, it is not possible to provide a unifying declaration about the nature of these osmolytes. Therefore, a complete acquaintance of each osmolyte with respect to each protein is one of the most challenging tasks in the development of protein formulation and may be still craving to administer protein formulations through injection only. Further investigations are obligatory to evade undesirable outcomes in biopharmaceutical formulations as well as in the human body. The in-depth knowledge through the literature survey has drawn the attention of many researchers toward the filling of this gap by analyzing the effects of the different methylamines and amino acids from dilute solution to the molar concentration range and to design proteins with enhanced stability by understanding the factors determining protein stability. Additionally, from the cell biology point of view, it is interesting to study proteins in highly crowded solutions to mimic the intracellular environments. The lack of comprehensive investigations over a broad range of these osmolytes concentrations in protein systems intrigues our interest in this research field. It is a particularly appealing task to shed light on the mechanism of stabilization/destabilization of the protein in the presence of various methylamines and amino acids. In this context, we have investigated the interactions of methylamines and amino acids with a protease enzyme, stem bromelain (BM), which play a central role in determining the native conformation of BM at its maximum activity. The chosen osmolytes are betaine, sarcosine, proline, and arginine. The sarcosine and betaine are homologous zwitterions at neutral pH with increased methylation of amine group in betaine as compared to sarcosine.10,29 Proline is one of the best studied amino acid according to literature survey,30 whereas arginine is well argued amino acid with respect to its stabilizing or destabilizing nature.24,31,32 There is no doubt that the effect of any osmolyte is protein-dependent in each protein formulation. To date, these cosolutes have not been studied for stability of BM which is quite important from applications point of view reported elsewhere.33 This meticulous study of interactions can be used to reveal the mystery of BM clinical utility and also to make a successful path from administration site to the target site if used as drug for a number of critical problems, such as inflammation, tumor, etc. To properly access the mechanism of stabilization of BM in the presence of betaine, sarcosine, proline, and arginine, we used fluorescence and UV−vis spectroscopy, circular dichro-

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MATERIALS AND METHODS Materials. Bromelain (E. C. 3.4.22.32) lot No. B4882 from Ananas cosmosus, betaine, sarcosine, arginine, proline, and 1anilinonaphthalene-8-sulfonic acid (ANS) were purchased from Sigma-Aldrich, USA. Anhydrous sodium phosphate monobasic and sodium phosphate dibasic dihydrate were purchased from Sisco Research Lab (SRL), India. For proteolytic activity study, casein (Hammarsten), sodium acetate, and acetic acid were also taken from the SRL, India. All other chemicals used were of analytical grade with high purity. Sample Preparation. The enzyme samples were prepared in 10 mM sodium phosphate buffer at pH 7 with 0.5 mg/mL enzyme concentration for all measurements. For DLS measurement, an enzyme concentration of 1 mg/mL was used. ANS solution was prepared at 1 mg/1 mL in buffer and 5 μL of this solution was added in each sample during ANS fluorescence study. For all gravimetric measurements, AND (Japan) balance with a precision of ±0.00001 g was used. Distilled deionized water with resistivity of 18.3 Ωcm was used to prepare all samples. After completely dissolving the enzyme in the solution, the mixture was filtered with a 0.22 μm disposal filter (Millipore, Millex-GS) through a syringe and were incubated for 1 h at 25 °C in order to obtain complete equilibrium before performing experiments. The stability and activity of BM were studied in the presence and absence of 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 M of all osmolytes for all experiments except arginine. Due to the solubility issue, arginine was used until 1 M. Steady State Fluorescence Measurements of BM in Varying Concentrations of Osmolytes. Steady state fluorescence emission spectra measurements were performed at 25 °C in Cary Eclipse fluorescence spectrofluorimeter (Varian optical spectroscopy instruments, Mulgrave, Victoria, Australia) equipped with an intense xenon flash lamp and a Peltier-type temperature controller with a precision of ±0.05 °C using the excitation wavelength at 295 nm. All emission spectra were recorded at a concentration 0.5 mg/mL of BM in the presence of a range of concentration of various osmolytes using a slit width for the excitation and emission at 5 and 10 nm, respectively, between 310 and 450 nm. All spectra were averaged for three scans. For an ANS binding fluorescence study, an excitation wavelength was set at 380 nm and emission spectra were taken from 400 to 550 nm. All other parameters were kept the same. Proper subtractions of blank solutions (i.e., without protein) were done from each sample for ANS binding studies because in some of the studied osmolytes, there was osmolyte-ANS binding at their higher concentrations. However, blank solutions of osmolytes showed λmax at a quite lower wavelength as compared to λmax for ANS binding to BM which did not interrupt the obtained results. Absorption Spectroscopy Analysis of BM in Varying Concentrations of Osmolytes. Absorption spectra for BM B


Article


Activity Measurements of BM in the Presence of Various Osmolytes. BM activity is assayed using a UV−vis spectrophotometer (UV-1800 Simadzu) providing denatured casein solution (0.5 mL of 0.5%) as a substrate which was incubated for 10 min at 25 °C with BM in buffer at pH 7.0 (0.5 mL of 0.5 mg/mL) pretreated with the various concentrations of methylamines and amino acids. After a reaction time of 10 min, 1 mL of trichloroacetic acid (TCA) solution (0.11 M of TCA, 0.22 M sodium acetate, and 0.33 M acetic acid), was used to stop the reaction and the precipitates of the undigested casein were removed by the centrifuging the samples at 10000 rpm for 10 min. The reaction product was correlated with the absorbance of a reagent blank measured spectrophotometrically at 275 nm. From the standard curve of the absorbance of known quantities of the Tyr, activity (units per mL) of BM samples which is the amount of the micromoles of Tyr equivalents released from casein per min, is calculated using the following equation:

in the absence and presence of various concentrations of osmolytes were recorded on a Shimadzu UV-1800 (Japan) spectrophotometer with the highest resolution (1 nm) using matched 1 cm path length quartz cuvettes at 25 °C. The blank solutions subtraction was done for each sample and obtained spectra were averaged for three scans. Spectral Characterization of BM in Various Osmolytes using Circular Dichroism. The circular dichroism (CD) spectral studies were carried out for BM as a function of concentration of osmolytes after pre-equilibration of all samples at 25 °C for 1 h. All CD spectra are monitored by using a PiStar-180 spectrophotometer (Applied Photophysics, U.K.) equipped with a Peltier system for temperature control with an accuracy of ±0.1 °C. The system is calibrated using (1S)-(+)-10-camphorsulfonic acid (Aldrich, Milwaukee, WI) which displays a molar extinction coefficient of 34.5 M cm−1 at 285 nm and molar ellipticity (Θ) of 2.36 M cm−1 at 295 nm. Far-UV and near-UV CD spectra are taken in the range 185−250 nm and 250−300 nm, respectively, by using a cuvette with path length of 0.1 and 1 cm, respectively, at a response time of 1 s and 1 nm bandwidth using a scan speed 50 nm/min. All spectra are averaged for three scans. All blank solutions were appropriately subtracted from all studied samples. Dynamic Light Scattering Measurement. The dynamic light scattering measurements were performed at a scattering angle of 90° by a Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) with He−Ne laser of 4 mW operating at a fixed wavelength, 633 nm, and emitting vertically polarized light. An inbuilt thermostatic sample chamber was used for maintaining temperature equal to 25 °C in an airtight quartz cell where a sample was loaded. The instrument detection range was from 0.1 nm to 10 μm. In order to avoid interference caused by the buffer, very dilute buffer (5 mM) at pH 7 is used to prepare all samples. All samples were filtered through a 0.1 μm pore size Millipore syringe filter. Specifically, DLS size distributions are obtained by analyzing the scattered light fluctuations, which are, in turn, related to the size of particle, viscosity and refractive index. Therefore, the values of refractive index, dielectric constant and viscosity were also taken into consideration for all samples. Each measurement consisted of three subsequent individual runs and averaged for three concordant reading. From the time dependent fluctuations in the scattering intensity of light, this instrument measured the translational a diffusion coefficient by using the instrumental software. The hydrodynamic diameter (dH) was attained from diffusion coefficient by means of a well-known Stokes− Einstein equation. The data were further evaluated by plotting using origin 9 software (OriginLab Corp., Northampton, MA, USA). Thermal Equilibrium Unfolding of BM in the Presence of Various Osmolytes. The cosolvents induced equilibrium unfolding studies of the BM were conducted over a temperature range from 15 to 95 °C, using Trp fluorescence as a probe in the same Cary Eclipse fluorescence spectrofluorimeter at a heating rate of 1 °C min−1 providing adequate time for equilibration. An excitation wavelength of 295 nm was used in order to avoid the radiation energy transfer from Tyr residues to Trp residues. All unfolding transitions of BM at pH 7.0 are analyzed by assuming the two state unfolding mechanism by following the method as described elsewhere.34,35

activity =

(μmole tyrosine equivalents released)(V ) (time of assay in minutes)(v1)(v2)

(1)

where V is total volume of assay in mL, v1 is volume of enzyme used in mL and v2 is volume of sample in mL used for UV measurements. MD Simulation Studies of BM in Water and in 1 M Osmolyte. The MD simulations of BM in water and in 1 M (arginine, sarcosine, betaine, and proline) were carried out using GROMACS version 4.5.5.36,37 The starting structure taken for MD simulations is a theoretical model of BM (PDBID: 1W0Q).38 The GROMOS 53a6 force field39 was used as the parameters for BM and cosolvents. The parameters of the cosolvents were taken from the Automated Topology Builder (ATB) database.40 The SPC/E model41 was used for water molecules. The BM enzyme was located in a cubic box containing1M (arginine, sarcosine betaine, and proline). The box was large enough to contain BM surrounded by 1 nm of solvent on all sides. The total charges of the simulation systems were neutralized with counterions. The molecular number of cosolvent was 202 molecules. The Newton’s equations of motion were performed with a MD Leap frog integrator42 using a time step of 2 fs. The chemical bonds were constrained with the LINCS algorithm. The cutoffs for the Coulomb interactions and Lennard-Jones interactions were 1.0 and 1.0 nm, respectively. The particle mesh Ewald (PME) algorithm43 was used to treat the electrostatic interactions. The simulation box was minimized by using the steepest descent algorithm. The temperature of the periodic box was maintained constant by the Nosé−Hoover thermostat44,45 and the pressure was fixed by the Parrinello−Rahman barostat.46 Each system was equilibrated for 1 ns with NVT ensemble at 300 K, and the protein bonds were restrained. The position restraints of the protein were finally released and the system was equilibrated for 100 ns (50 000 000 steps) at 300 K and 1 bar (NPT ensemble). The results of the simulation were visualized using the VMD program.47 The average number of hydrogen bonds per molecule for each saved MD trajectory frame, ⟨NHB⟩, was determined with a cut off donor−acceptor (DA) distance of 0.35 nm and a cut off acceptor−donor-hydrogen (DHA) angle of 30°.48 The radial distribution functions (RDFs) gAB(r) of i atom with respect to j atom were computed by C


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Figure 1. Fluorescence spectra analysis of the BM conformation in the presence of methylamines and amino acids at 25 °C: BM in the presence of buffer (black) and (a) betaine, (b) sarcosine, (c) arginine, and (d) proline as a function of concentrations, 0.1 M (red), 0.5 M (green), 1.0 M (blue), 1.5 M (cyan), 2.0 M (pink), 2.5 M (yellow), and 3.0 M (dark yellow). NA

NB

4πr 2gAB(r ) = V ∑ ∑ P(r ) iϵA jϵB

300 K and 1 bar (NPT ensemble). The simulated densities are (1036.03 ± 0.15, 1052.03 ± 0.12, 1030.84 ± 0.10, and 1026.55 ± 0.13) kg·m−3 for 1 M sarcosine, arginine, proline, and betaine; and their experimental densities using Anton paar DMA 4500 vibrating tube denstimeter are (1032.35, 1049.85, 1035.34, and 1022.74) kg·m−3, respectively. The deviations of the simulated densities from the measured ones are ranging from 0.2 to 0.4%. Several studies have been used the densities alone to validate the reliability of the force-fieldbased simulation.52−54 Therefore, the agreement between our simulated densities and the experimental values encourages us to be reasonably confident about our MD simulations.

(2)

where V is the volume and P(r) refers to the probability of finding a B atom at distance r from an A atom. The preferential binding and local preferential binding coefficient (Γ)49 and local bulk partition coefficient (Kp)50,51 are calculated in the NPT ensemble using the last 15 ns. Both Γ and Kp are computed as a function with distance from the BM surface and center of mass of osmolyte and water molecules, by using eqs 3 and 4. Γ=

Kp =

ns −

Nstot − ns × nw Nwtot − n w

⟨ns⟩Nwtot ⟨n w ⟩Nstot

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RESULTS AND DISCUSSION We investigate the biomolecular interactions of methylamines and amino acids with enzyme by applying a series of biophysical techniques and MD simulations for characterizing changes in the structural and thermal stability, conformational size, and proteolytic activity of the enzyme. The enzyme solutions containing different osmolytes exhibit overall diverse consequences which are determined by the physical and chemical properties of each osmolyte with respect to the protein under study. Moreover, higher concentrations of these osmolytes present a different solvent environment than do the dilute solutions, thereby; some aspects of the properties must be somewhat different at different concentrations. At this point, it seems pertinent to declare the complete assessment of all the experimental and simulations results by considering

(3)

(4)

where ⟨ns⟩ and ⟨nw⟩ are the average number of molecules of osmolye and water in the simulated system, respectively. Ntot w and Ntot s are the total number of osmolyte and water in the simulated system, respectively. In order to validate the employed force field for the studied osmolytes, we have computed the density of 1 M arginine, sarcosine, proline or betaine and compared with the experimental densities. The simulated binary mixtures were consisted of 202 sarcosine + 9864 water, 202 arginine + 9181 water, 202 proline + 9579 water, and 202 betaine + 9492 water molecules. These mixtures were simulated for 20 ns at D


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Figure 2. UV−vis spectral analysis of BM at 25 °C: BM in the presence of buffer (black) and (a) betaine, (b) sarcosine, (c) arginine, and (d) proline as a function of concentrations, 0.1 M (red), 0.5 M (green), 1.0 M (blue), 1.5 M (cyan), 2.0 M (pink), 2.5 M (yellow), and 3.0 M (dark yellow).

Figure 1c) escorted with decrease in Imax as compared to BM in buffer depicting more exposure of Trp to the polar environment. Figure 2 symbolizes the absorbance of BM in the presence of betaine, sarcosine, arginine, and proline. BM in buffer (control) exhibits an absorbance maximum (Amax) at 280 nm. In Figure 2a, b, c, and d, there is a decrease in the absorbance of BM with increasing concentration of betaine, sarcosine, arginine, and proline, respectively, indicating changes in the microenvironment of aromatic residues of BM. Amplified absorbance indicates more exposure of aromatic residues and declined absorbance depicts internalization of these residues.55 However, as arginine is highly polar as compared to water and may strongly bind to the aromatic residues, thereby, exposure of aromatic residues might be causing a sharp decrease in the absorbance as compared to that of BM in buffer (Figure 2c).56 Figure 3 comprises of ANS fluorescence of BM which provides information about the changes in the hydrophobic core of BM. From the increased ANS fluorescence in Figure 3c, it may be assumed here that the hydrophobic region in BM is accessible to ANS to more extent as a function of concentrations of arginine as compared to that of BM in buffer. The results also show a paramount blue shift of 7 nm (λmax for BM in buffer = 487 nm and λmax for BM in 1 M arginine = 480 nm). Therefore, it can be inferred here that hydrophobic interactions are deteriorated to an extensive degree in the presence of arginine.

the reasonable selection of these osmolytes in the present work and also rationalizing the available open literature. Predicting Structural Changes in BM by Intrinsic Fluorescence, ANS Fluorescence, UV−vis, and CD Spectroscopy. In order to evaluate the structural changes and also to access the folding properties of BM as a function of varying concentrations of osmolytes, the changes in the tryptophan (Trp) environment can be studied. Therefore, we employed the use of intrinsic fluorescence of Trp at room temperature. Figure 1 represents the intrinsic fluorescence of BM in the presence of betaine, sarcosine, arginine, and proline. BM in buffer (black spectra in Figure 1) displays a fluorescence intensity maximum (Imax) at 348 nm. In Figure 1a, it is shown that with increase in the concentrations of betaine until 3 M, there is a continuous increase in the Imax for BM accompanied by a blue shift (from 348 to 344 nm) as compared to that in buffer which may be attributed to the decrease in the exposure of Trp to the polar environment, thereby, reduced solvent quenching. For BM in sarcosine and proline (Figure 1b and d), there is no λmax shift at very low concentrations (until 1.0 M for sarcosine and 1.5 M proline). Thereafter, a significant blue shift in λmax of BM is detected with increase in concentrations of sarcosine and proline as compared to that of BM in buffer (from 348 to 345 nm) which indicates internalization of Trp toward hydrophobic core where may be some charged residue quenchers resulting in decreased Imax (Figure 1b and d). On the contrary, in case of increasing concentration of arginine, there is significant amount of red shift (from 348 to 353 nm in E


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Figure 3. ANS fluorescence spectra analysis of the BM conformation in the presence of methylamines and amino acids at 25 °C: BM in the presence of buffer (black) and (a) betaine, (b) sarcosine, (c) arginine, and (d) proline as a function of concentrations, 0.1 M (red), 0.5 M (green), 1.0 M (blue), 1.5 M (cyan), 2.0 M (pink), 2.5 M (yellow), and 3.0 M (dark yellow).

β-strands, then to other structures, such as β-turns which is obvious from the appearance of a negative band in the range of 185−193 nm). β-turn and other structures are increased as a compensation of β-sheets. At very higher concentrations, βturn also decreases leading to the overall highly decreased secondary structures as is clear from decreased ellipticity of the negative band at ∼220−229 nm and also from shifting of the positive band toward higher wavelength. On the other hand, there is abrupt decrease in the negative ellipticity values of BM on addition of increasing concentrations of arginine and proline (Figures 4c and d). Thereby, it is evident here that the secondary structures of BM are highly diminished by the use of arginine and proline. It is mandatory to state here that the data for secondary structures of BM in arginine and proline (Figures 4c and d) may not be absolutely reliable because subtraction of blank samples (without BM) with very high absorbance of arginine and proline in the far-UV region may lead to some disagreement. Figure 5 elucidates near-UV CD spectra which reveal the environment of the aromatic amino acid residues (mainly Trp, Tyr, and Phe) and gives information about the tertiary structure of BM in the presence of osmolytes. As can be seen from Figure 5, there are CD positive bands around 256, 270, and 280 nm. The CD bands from individual residues may be positive or negative and may vary widely in intensity, therefore, it is often difficult to separate out the contributions of individual aromatic residues. Consequently, all spectra have been explicated in a broad way concluding from overall

On the other hand, Figure 3b and d, respectively, representing BM in changing concentration of sarcosine and proline indicate noteworthy red shift (λmax is 494 nm for 3 M sarcosine and 493 nm for 3 M proline) with decreasing intensity as compared to that of control that may portray the burial of hydrophobes into the core of BM. The ANS fluorescence intensity of BM in betaine is decreased (Figure 3a), however, not to a very good extent which depict that hydrophobic pockets are not perturbed largely as compared to other osmolytes. In order to confirm whether these conformational changes in the Trp environment are accompanied by changes in the secondary and tertiary structures of the enzyme, we further employed far UV-CD and near UV-CD spectroscopy, respectively. A black spectrum (Figure 4) represents BM in buffer possessing three bands; one sharp negative band at ∼208 nm, second negative shallow band at ∼222 nm, and a positive band at ∼190 nm, which are known to be typical for protein containing α + β characteristics, furthermore, found to be consistent with results by Reyna et al.57 In Figures 4a and b, decreased negative ellipticites, especially, of the band at 208 nm can be clearly speculated with increasing concentration of betaine and sarcosine in comparison to that of BM in buffer, correspondingly, which may be pointed to the decreased secondary structures, mainly α-structures. The α-structures may have been transformed into some other structures which are quite apparent from band position in Figures 4a and b (shifting of 208 nm band toward 214 nm, a characteristic of F


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Figure 4. Far-UV CD analysis of BM conformation in the presence of the methylamines and amino acids at 25 °C: BM in the presence of buffer (black) and (a) betaine, (b) sarcosine, (c) arginine, and (d) proline as a function of concentrations, 0.1 M (red), 0.5 M (green), 1.0 M (blue), 1.5 M (cyan), 2.0 M (pink), 2.5 M (yellow), and 3.0 M (dark yellow).

changes in the positive ellipticity not particularly specifying the environment of three of aromatic amino acid residues (Trp, Tyr, and Phe) and disulfide bonds. With increase in the concentration of all of four cosolvents, the noticed decrease in positive ellipticities is implying, here, that the aromatic residues are moving toward a less asymmetric environment as a result of loosening of some of the tertiary contacts (Figures 5a−d). However, the tertiary structure is not disrupted to a great extent even at high concentrations of osmolytes except in case of arginine as is clear from ellipticities values in Figure 5a, b, and d. Interestingly, at higher concentration of proline, tertiary structures are appreciably enhanced even more than those in buffer as is clear from increased positive ellipticities at 2.5 and 3.0 M (Figure 5d). On the other hand, arginine is comparatively found to be more effective in disrupting the tertiary structure of BM even at its lower concentration (Figure 5c). Furthermore, the ellipticity values are even changed to negative at 1 M of arginine, thereby, depicting highly disturbed tertiary structure of BM. Conformational Size of BM in the Presence of Methylamines and Amino Acids by DLS Measurements. The DLS data provide further insight into the mechanism of interaction between BM and cosolvents and verify the above proposed explanation for the observed conformational changes in BM. In order to estimate the size of the BM in the presence of varying concentrations of different osmolytes, all investigated samples were analyzed by DLS.

Figure 6 demonstrates intensity distribution graph of BM in buffer and in the presence of varying concentrations of betaine, sarcosine, arginine and proline. All peaks below 1 nm attributing to solvent/cosolvent are ignored. The numerical values of hydrodynamic diameter (dH) obtained for all samples are also summarized in Table 1. As is clear from Figure 6, the peak for BM in buffer is very monodisperse (shown black in Figure 6). Polydispersity of BM in all samples also does not surpass 20%. The dH for BM in buffer (control) is ∼4.7 nm that is in good agreement with the approximated value (∼4.6 nm) on the basis of molecular weight and number of amino acid residues from a Zetasizer software. From dH profile in Table 1, the dH of BM in varying concentrations of betaine, sarcosine, and proline is found to be decreasing from ∼4.7 to ∼3.8 nm. Consequently, it is evident that the BM structure in the presence of these osmolyte solutions is somewhat compact in comparison to BM in buffer (Figure 6a, b, and d). However, arginine affected dH to a very large extent depicting highly denatured state with open structure. The dH changes drastically from ∼4.7 (control) to ∼27, 39, and 44 nm at 0.1, 0.5, and 1 M of arginine, respectively. With an exception of arginine, the decrements in dH for BM in these cosolvents are accounting for stabilizing effects on BM structure. Arginine is quite effective in expanding BM structure and also its clusters associated with the BM surface which may attribute to the unexpectedly large in increase dH of BM. According to a simulation study,32 the extension of protein surface by associated arginine molecules may account G


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Figure 5. Near-UV CD analysis of BM conformation in the presence of the methylamines and amino acids at 25 °C: BM in the presence of buffer (black) and (a) betaine, (b) sarcosine, (c) arginine, and (d) proline as a function of concentrations, 0.1 M (red), 0.5 M (green), 1.0 M (blue), 1.5 M (cyan), 2.0 M (pink), 2.5 M (yellow), and 3.0 M (dark yellow).

65.0 °C for arginine and 65.4, 65.1, and 64.6 °C for sarcosine at 0.1, 0.5, and 1.0 M, respectively. At 1 M of each osmolyte, the order of efficacy to increase the thermal stability of BM is as proline > betaine > buffer > arginine > sarcosine. Moreover, the stabilizing effects of proline and betaine are there or thereabouts analogous to each other as can be considered from Figure 7. The discrepancy in arginine and sarcosine at lower concentrations may be due to comparatively strong binding efficiency of these molecules to the BM surface, especially through more H-bonding which may lead to reduction in the thermal stability of BM. These strong preferential interactions by these osmolytes associated at the protein surface prevail over its preferential exclusion from rest of the protein surface. This type of explanation is also reported by Vagenende et al.58 for lysozyme in arginine solution where preferential interaction coefficient is appreciably negative up to 1 M of arginine. The thermal stabilization/destabilization by methylamines and amino acids is commonly considered as a consequence of contribution from different types of interactions. Evaluation of Activation/Inhibition of BM by Enzyme Assay. To correlate BM stability with activity in the presence of osmolyte, the activity assay for BM with casein as a substrate was performed in the absence and presence of

for major fraction of observed increase in the apparent protein diameter which is completely based on the observation of their long residence times (∼50 ns) at protein surface loci during simulation. Ultimately, it can also be clearly revealed from Figure 6 that in the presence of varying concentration of methylamines and amino acids, aggregates size of BM (>100 nm) is altered to a large extent and found to be reduced which may indicate that all studied osmolytes are inhibitors of aggregates of BM. Thermal Stabilization of BM in the Presence of Methylamines and Amino Acids. Furthermore, with the intention of a relation between structural stability and thermal stability, the thermal fluorescence study was employed. A different scenario comes from analysis of the thermal stability of BM in the presence of these methylamines and amino acids. The transition temperature (Tm) is an essential parameter for thermal stability of an enzyme. The Tm for BM in buffer (control) is ∼65.5 °C (Figure 7). The increasing Tm values clearly reveal a significant stabilizing effect by proline and betaine in comparison to the control. On the contrary, as can be evidently speculated from Figure 7, Tm values of BM in arginine and sarcosine until 1 M are more or less similar to BM in buffer, afterward, sarcosine increases BM thermal stability (arginine is not soluble at concentration more than 1 M). The Tm values for BM are 65.8, 65.3, and H


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Figure 6. Hydrodynamic diameter (dH) of BM at 25 °C: BM in the presence of buffer (black) and (a) betaine, (b) sarcosine, (c) arginine, and (d) proline as a function of concentrations, 0.1 M (red), 0.5 M (green), 1.0 M (blue), 1.5 M (cyan), 2.0 M (pink), 2.5 M (yellow), and 3.0 M (dark yellow).

Table 1. Hydrodynamic Diameters (dH) of BM in the Presence of Various Osmolytes conc. of osmolytes [M] 0.0 0.1 0.5 1.0 1.5 2.0 2.5 3.0

betaine 4.67 4.67 4.45 4.45 4.20 4.00 3.85 3.80

± ± ± ± ± ± ± ±

0.2 0.3 0.3 0.2 0.2 0.1 0.4 0.2

sarcosine

arginine

± ± ± ± ± ± ± ±

4.67 ± 0.2 27.12 ± 0.3 39.4 ± 0.3 44.4 ± 0.4

4.67 4.67 4.67 4.45 4.35 4.25 4.06 3.88

0.2 0.3 0.3 0.2 0.3 0.2 0.2 0.1

proline 4.67 4.67 4.45 4.45 4.24 4.05 3.88 3.85

± ± ± ± ± ± ± ±

0.2 0.3 0.1 0.1 0.1 0.1 0.2 0.2

Figure 7. Variation in Tm values of BM in buffer (black) and in varying concentrations of betaine (red), sarcosine (green), arginine (blue), and proline (cyan). All values are averaged for three concordant readings. The error in Tm does not exceed 0.1 °C.

different osmolytes. Figure 8 displays the percentage activity of BM in various osmolytes. It can be undoubtedly witnessed in Figure 8 that the caseinolytic activity of BM remains more or less analogous to that of BM in buffer (control) up to 0.5 M of betaine, sarcosine, and proline. After that, there is regular decrease in the percentage activity of BM in all of the three cases except at 1 M proline. Arginine inactivated BM to an extreme extent even at very low concentration (0.1 M) and further increase in its concentration leads to comparatively more activity, however, still lesser than control. It is interesting to note that proline at 1 M exhibits maximum activity among all studied osmolytes followed by betaine, arginine, and sarcosine in decreasing order.

From the obtained activity, it is pretty evident that there is a varying degree of inhibition of BM activity by these additives. It is, however, reasonably difficult to suggest the root behind these acquired results, i.e., whether it is related to decreased substrate binding or catalytic site functionality or both. There may also be the probability of the contribution from the changes in the surface properties of the enzyme upon addition of these additives. Arginine may be leading to I


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Figure 8. Proteolytic activity measurements of BM in absence and presence of methylamines and amino acids at 25 °C: The variation in percentage activity of BM in buffer (black) and in varying concentrations of betaine (red), sarcosine (green), arginine (blue), and proline (cyan). All values are averaged for three concordant readings. Error in percentage activity is not more than 5%.

Figure 9. Plot of the average hydrogen bond < NHB> between BM and osmolytes as a function of simulation run length.

conditions. The precise explanation behind the decline in the activity of BM by different osmolytes can be well afforded by the MD simulations MD Simulation Studies of BM in Water and in 1 M of Various Osmolytes. The electrostatic (Coulomb, Ecoul) and the van der Waals (Lennard-Jones, ELJ) interaction, and total interaction (Eint = Ecoul+ELJ) energies obtained from the MD simulations of BM in 1 M (arginine, sarcosine, proline, and betaine) are reported in Table 2. The interaction energy (Eint) values between BM and osmolytes are decreased in order: sarcosine > arginine > proline > betaine. This interaction has reduced the intramolecular interactions of BM, as it can be seen from the reverse order in intramolecular protein−protein interaction energy (Eint), betaine > proline > arginine > sarcosine. It is clear from Table 2 that these osmolytes mainly interact with BM through electrostatic force. This is to be expected since these compounds exist in zwitterionic form at the physiological pH. Figure 9 shows the average hydrogen bonds < NHB> between BM and cosolvents, revealing the same order of Eint except for arginine and sarcosine. Arginine was found to form greater hydrogen bonds than that of sarcosine. This is probably due to that the former has more hydrogen bond donors compared with the latter. The interaction energy (Eint) between BM and water is decreasing in the order of betaine ≈ proline > sarcosine > arginine, indicating that BM gets more hydrated in the presence of betaine and proline than those in sarcosine and arginine solutions. Thus, the high intraprotein interaction and high hydrated state of BM in betaine and proline solutions as compared in those in sarcosine and arginine solutions are probably responsible for the high thermal stability of BM in betaine/ proline solutions than that in arginine/sarcosine solutions. These simulation results show that efficacies of proline and betaine to influence the stability of BM are not varying from each other to an appreciable extent. For a further interpretation of the thermal stability of BM, we focus on the preferential binding and exclusion of osmolytes. Such information should be obtained from both preferential binding coefficient (Γ) and local bulk partition coefficient (Kp). Figure 10a shows Kp of osmolytes as a function of the distance (r) from protein surface. If Kp greater than 1, the osmolyte molecules accumulate in vicinity of the protein surface, Kp< 1 if they are depleted. As one can see in this figure, the local-bulk partition coefficient is greater than 1 in all cases, indicating that osmolytes strongly bend to protein.

Table 2. Average Electrostatic (Coulomb), Ecoul, and the van der Waals (Lennard-Jones), ELJ, Energies Obtained from the MD Simulations, Based on Short Range Energy Components interactions water protein−protein protein−water arginine protein−protein protein- water protein−ligand betaine protein−protein protein- water protein- ligand proline protein−protein protein−water protein- ligand sarcosine protein−protein protein- water protein- ligand a

ECoul (kJ/mol)

err. est.

ELJ (kJ/mol)

err. est.

−9441 −13027

64 200

−5249 −791

38 26

−14690 −13818

−8982.4 −10886 −3177

37 460 400

−5187 −548 −905

31 37 89

−14169.4 −11434 −4082

−9305 −11116 −1925

51 230 72

−5102 −674 −900

13 10 40

−14407 −11790 −2825

−9323 −11122 −2082

48 190 79

−5064 −593 −1077

17 19 33

−14387 −11715 −3159

−8749 −10928 −3438

13 180 170

−4978 −612 −1223

5 7 46

−13727 −11540 −4661

Einta

Eint= Ecoul + ELJ

the greatest disruption of the conformation of BM, especially at the active site, resulting extremely diminished activity. It has been reviewed in the literature31 that arginine affects the substrate binding to the active site. In comparison to the case in 0.1 M of arginine, this type of disturbances is fairly decreased at 1 M arginine which may be due to removal of some arginine molecules from the surface which depicts relatively more activity. On the other hand, proline may be escorting more activity until 1 M due to compacting of BM that may cause a decrease in the distance between the active site residues than that of the control, ensuing more activity. Subsequently, higher concentrations of proline (>1 M) may cause more compactness of BM resulting BM active site toward more rigidity that is unable to work in a concerted manner which may account the decreased activity at these J


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Figure 10. Local-bulk partition coefficients (a) and preferential binding coefficients (b) as a function of distance to BM surface for sarcosine (black), arginine (red), proline (green), and betaine (blue), as obtained from MD simulations.

BM and osmolytes as reflected by the decreases in the activity of BM. It has been known that the deprotonation of the thiol group of Cys 25 in the BM’s active site by the adjacent amino acid residue, His 157, acts simultaneously in the hydrolysis of substrate.61,62 The distance between these two residues plays an important role in the catalytic activity of BM; in particular, the distance between the thiol proton and imidazole nitrogen atom. The increase in this distance leads to a decrease in the BM activity. Radial distribution functions (RDFs) for distances between thiol proton and imidazole nitrogen atoms obtained from the MD simulation results are plotted in Figure 11. The distances between these two atoms were found to be (1.6, 1.9, 4.3, 5.0, and 6.2) Å, for proline, water, betaine, arginine, and sarcosine, respectively which obtained from the peaks position. Similar trend (proline > water > betaine > arginine > sarcosine) for the BM activity in 1 M osmolyte was obtained from experimental results which is emphasizing the role of these distances in the catalytic activity of BM. It is interesting to observe that the distance between thiol’s proton and imidazole’s nitrogen atoms is smaller than that in water which explains the higher activity of BM in 1 M proline as compared to water. Finally, analysis of the hydrogen bond formation between catalytic residues and the studied cosolvents will shed light on the reason behind the change in distance between catalytic residues. Sarcosine, arginine, betaine are found to form hydrogen bonds with the catalytic residue while proline is not. The time averaged number of hydrogen bonds between the catalytic residues and osmolytes are 2.40, 2.32, and 1.48 for sarcosine, arginine, and betaine, respectively. Figure 12 presents MD snapshots displaying the hydrogen bonds between catalytic residues and osmolytes. The carboxylic oxygen atoms of arginine form hydrogen bonds with the thiol’s hydrogen and imidazole’s polar hydrogen atoms as well as with the amide hydrogen atom of Cys25. Two sarcosine molecules form two hydrogen bonds with the catalytic sites. The carboxylic oxygen atom of one molecule forms hydrogen bond with the thiol proton and the carboxylic oxygen atom of the other molecule forms hydrogen bond with the imidazole polar hydrogen atom. The carboxylic oxygens of betaine are found to form hydrogen bonds with the thiol hydrogen and the amide hydrogen atoms of Cys 25. It can be seen from Figure 12 that the way that the osmolytes (sarcosine, arginine

Figure 11. RDFs for distances between the thiol proton and imidazole nitrogen atoms for sarcosine (black), arginine (red), proline (green), betaine (blue), and water (cyan) as obtained from MD simulations.

This should not be surprising since we were able to observe direct contacts between the osmolytes and protein in terms of intermolecular hydrogen bonds. However, the trend in Kp of the osmolytes follows the order sarcosine > arginine > proline > betaine. The first sarcosine accumulation shell occurs at a distance of r = 0.35 nm, while for other osmolytes are shown at r = 0.5 nm. Therefore, arginine, proline, and betaine are more excluded from the protein surface when compared to sarcosine. The preferential binding coefficients of BM in the osmolyte solutions are given in Figure 10b. Γ may have positive or negative values, suggesting that the interaction between the protein and osmolytes are favorable or unfavorable, respectively. Not surprisingly, Γ is positive for all osmolytes which is matching with Kp greater than 1 and similar trend as for Kp is obtained. Additionally, preferential exclusion of arginine, proline, and betaine is observed at (r ≈ 0.3 nm), and this effect is related to a presence of a thin water layer in direct contact with protein surface.59 Whereas sarcosine is insignificantly excluded at (r ≈ 0.25 nm). The Γ and Kp results suggest that the studied osmolytes are preferentially binding with the protein surface and this contradicts the popular view that osmolytes are preferentially excluded from the protein surface.60 However, this should not be surprising because we found a strong interactions between K


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Figure 12. Snapshots (at 100 ns) showing the hydrogen bond formation between the catalytic residues and osmolytes obtained from the MD simulations of BM in 1 M of (a) arginine, (b) sarcosine, and (c) betaine; (d) the catalytic pocket in 1 M proline.

Arg, His, or the N−H group of the peptide bond as well as through some indirect interactions.9,26 With increase in the concentration of betaine, there may be no more water molecules available to hydrate betaine molecules and less water is there for competition with betaine for the protein surface.63 Moreover, the interactions between betaine with the side chains is also supported by the MD data in Table 2. In addition, the strong interactions of BM−betaine attribute to the decreased intramolecular interactions of the BM which is quite obvious from Figure 4a indicating intramolecular H-bonding. The changed conformation of BM may be positioning Trp in a situation that Trp fluorescence is less quenched and contributes to increased fluorescence intensity and decreased absorbance in Figures 1a and 2a, respectively. The increased hydrophobic interactions may be responsible for the increased stability as compared to control which is concluded from the blue shift in Trp fluorescence, enhanced ANS fluorescence, and decreased dH of BM. The above-discussed side chain interactions are more favorable in sarcosine as compared to the betaine as is clear from our simulation data (Table 2). Additionally, simulation results show that BM is more hydrated in betaine as compared to that in sarcosine. Therefore, sarcosine is preferentially more binding to protein surface as compared to betaine. Consequently, intramolecular H-bonding interactions of BM are decreased (Figure 4b). However, sarcosine

and betaine) bind to the catalytic residues was increased the distance between the thiol proton and imidazole nitrogen atom. Proline does not form hydrogen bonds with the catalytic residues. Therefore, the decrease in the activity of BM in the presence of the studied cosolvents is due to the hydrogen-bonding formation between the cosolvent and the catalytic active site. To justify all the results mentioned above, it is necessary to discuss the nature of each osmolyte with respect to itself, water and protein. Betaine, sarcosine, arginine and proline are of zwitterionic nature at physiological pH and possess large dipole moments; hence, a strong binding tendency with water.63 However, BM behaves in a dissimilar way in these four osmolytes depending on their different binding tendencies to the BM surface, water and itself. Betaine is highly hydrated in the ternary solution of betaine, water, and BM.63 There is partial positive charge on the methyl groups of betaine due to which methyl groups may be not too hydrophobic in nature and also interact with the water molecules. However, this effect is not as in the case of TMAO.64,65 Furthermore, due to charge−π interactions, betaine favorably interacts with the aromatic residues (Trp and Phe) of BM.9,26 Besides, BM possesses net positive charge on its surface at physiological pH (pI of BM > 7), thereby, more betaine molecules may favorably bind to the surface of the protein through direct interactions with the Lys, L


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of BM in buffer. At these high concentrations, proline aggregates formation by stacking ability through the pyrrolidine ring may also contributes to more preferential exclusion, in turn, more burial of hydrophobic groups in the protein core.69−71 As a consequence, tertiary structure of BM is increased at high concentration of proline (Figure 5d). However, no observation signifies binding of proline to the hydrophobic surface of the protein. MD simulation results also depict preferential hydration of BM in proline as compared to other osmolytes. Considering all the results, it must be expected that the overall effects are determined by the properties of each osmolyte and protein under study. A mechanism for cosolvent effects on the protein stability can be recommended in consistency with the recent advances on the osmolyte in the literature.72−74 Apparently, these results will facilitate to precisely model intrainter protein interactions in the presence of betaine, sarcosine, arginine, and proline. Both experimental and simulation studies put emphasis on the increased preferential interactions of arginine and sarcosine are responsible for their inabilities to stabilize the BM structure and thermal stability. The present study will possibly make a ground for various pharmaceutical applications of BM in vitro and in vivo as well.

may be causing increase in hydrophobic interactions in BM which is supported by blue shift in Trp fluorescence, decreased absorbance and decreased ANS fluorescence intensity, as shown in Figures 1b, 2b, and 3b and possibly will also contribute to decrease in BM size (Figure 6b). These interactions may be responsible for increase in the thermal stability of BM as a function of sarcosine concentrations. However, the binding interaction of sarcosine and BM may dominant at lower concentrations of sarcosine as explained by the MD simulations results and may be responsible for decreased thermal stability of BM at these conditions as compared to that of BM in buffer. In case of arginine, its guanidinium group (Gdm+) preferentially interacts with hydrophobic surfaces, in particular, with aromatic groups through van der Waals and cation−π interactions which may lead to a decrease in hydrophobic interactions.31,59,66 Rather than this, arginine also exhibits H-bonding and electrostatic interactions.58,66 In addition, Gdm+ interacts the protein carboxyl groups. At the studied concentrations of arginine, there may be efficient preferential binding of arginine to the protein surface which may cause a decrease in hydrophobic interactions in the protein (increased ANS fluorescence intensity, blue shift in Figure 3c, increased dH in Figure 6c and Table 2). The Trp environment is considerably changed and Trp get exposed to the solvent resulting red shift in Trp fluorescence (Figures 1c). Strong binding of arginine to protein surface diminishes both the secondary and tertiary structure of BM to an extremely appreciable extent (Figures 4c and 5c). With increasing concentration of arginine, the extent of selfinteractions of arginine molecules may enhance.58,66 Moreover, arginine-arginine interactions are stronger as compared to arginine-water interactions, consequently, Arg+ clusters start to form at the surface of the protein as well as in the bulk of solution. However, arginine molecules already bound to the surface of the protein favor the binding to other arginine molecules to form clusters. At 1 M of arginine, all the binding sites for arginine may become saturated and arginine molecules present in bulk may start to draw arginine from protein into bulk which may be responsible for less diminished activity at 1 M as compared to 0.1 M arginine. Proline undergoes strong H-bonding with water in comparison to water with itself that may result in structuring of water, in turn, preferential hydration of BM.67,68 The preferential hydration of BM may be accompanied by increased hydrophobic interaction which is clear from Figure 3d. The ANS fluorescence intensity is found to be decreased along with red shift for BM in proline in Figure 3d which indicates the burial of hydrophobes as a result of increased hydrophobic interactions. The burial of Trp is escorted by the decreased Trp fluorescence intensity of BM that gesticulate increased quenching of BM by charged residue quenchers (Figure 1d). However, proline escorts the lesser amount of change in the Trp environment at its lower concentration as is obvious from no change in λmax and minor decrease in fluorescence intensity (Figure 1d). Again, the scrutinized diminishment in absorbance of BM in Figure 2d may be owing to internalization of hydrophobic residues to the protein core (also can be seen in Figure 3d). Additionally, the enhanced water−BM interactions may attribute to the decreased intramolecular H-bonding. At higher concentrations of proline, the decrease in fluorescence intensity is paramount with a blue shift of 3 nm at 3 M proline as compared to that

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CONCLUSIONS This study reports denaturation and deactivation of BM by the use of methylamines and amino acids for the first time and provides a molecular level picture behind it. The enhanced preferential binding of sarcosine and arginine with BM are accountable for decreased thermal stability whereas, in comparison, the preferential hydration of BM in betaine and proline escorts increase in the thermal stability. Proline possesses the utmost efficacy to increase the thermal stability among all studied cosolvents attributing to highest preferential exclusion. It can be concluded here that direct H-bonding of cosolvent to the active site of BM is the leading factor for a decrease in the activity of BM except proline. Combined properties of both enzyme and cosolvent play an important role in the overall effects of methylamines and amino acids on BM. DLS results show that all cosolvents decrease aggregation in BM. This type of research is speculative at present and further research is needed to establish the place of these tests in the clinical laboratories. It is likely that the demand of the methylamines comprising systems will increase in the next few years.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. ac.in; Tel: +91-11-27666646-142; Fax: +91-11-2766 6605. ORCID

Pannuru Venkatesu: 0000-0002-8926-2861 Ming- Jer Lee: 0000-0001-7586-7379 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the support from the Department of Biotechnology (DBT), New Delhi, through the grant ref./file no. BT/PR5287/BRB/10/1068/2012 for financial support M


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and A.R. is grateful to University Grants Commission (UGC), New Delhi for providing SRF (Senior Research Fellowship).

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