A Spectroscopic Insight on Ethanol Induced Aggregation of Papain

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

A Spectroscopic Insight on Ethanol Induced Aggregation of Papain Vaisakh Mohan, Nilimesh Das, Aritra Das, Vipin Mishra, and Pratik Sen J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b12063 • Publication Date (Web): 18 Feb 2019 Downloaded from http://pubs.acs.org on February 18, 2019

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The Journal of Physical Chemistry

A Spectroscopic Insight on Ethanol Induced Aggregation of Papain Vaisakh Mohan, Nilimesh Das, Aritra Das, Vipin Mishra and Pratik Sen* Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur – 208 016, UP, India Abstract In this contribution, the structural and dynamical changes occurring to papain in ethanol-water binary solvent mixture have been investigated and compared with its denatured state. Steadystate fluorescence, solvation dynamics, time-resolved rotational anisotropy, circular dichroism and single molecular level fluorescence correlation spectroscopic studies were performed for this purpose. In ethanol-water mixture with XEtOH = 0.6, DACIA-tagged papain was found to undergo a blue shift of 12 nm, while in presence of 5 M GnHCl, a red shift of 5 nm was observed. Solvation dynamics of the system was also found to be different in presence of these external agents. In ethanol-water mixture the average solvation time was found to increase almost 2 fold as compared to that in water, while in presence of GnHCl only a marginal increase could be observed. These changes of DACIA-tagged papain in ethanolwater mixture is attributed to the aggregation of the protein in the presence of ethanol. The residual anisotropy was found to increase 14 fold and the rotational time component corresponding to the rotation of the probe molecule was found to increase by 4 fold in the ethanol-water mixture also gives a notion of the papain aggregation. Atomic force microscopy (AFM) confirms this aggregate formation, which is also quantified by fluorescence correlation spectroscopic (FCS) study. The hydrodynamic radius of the protein aggregates in ethanol-water mixture was calculated to be ~155 Å as compared to the corresponding value of 18.4 Å in the case of native monomer papain. Also, it confirmed that the aggregate formation takes place even in nanomolar concentration of papain. Analysis of circular dichroism spectra of papain showed that an increase in the β-sheet content of papain at the expense of α-helix and the random coil with an increase of ethanol mole fraction may be responsible for this aggregation process.

* Corresponding author Dr. Pratik Sen, Department of Chemistry, IIT Kanpur E-mail: [email protected], Fax: +91-512-259-6806

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Introduction It has been long known that alcohol-water mixtures show interesting anomalies in many thermodynamic and physiochemical properties including partial molar volume, viscosity, compressibility and diffusion coefficient.1–8 Many experimental techniques and theoretical methods have been used to understand these anomalous behaviour including Raman spectroscopy, mass spectroscopy and molecular dynamics simulation.9–18 The effect of these alcohol-water mixtures on proteins and other macromolecules of biological importance has also been widely studied.19–34 Bhattacharya and co-workers have looked at the effect of ethanol-water mixture on lysozyme, where they found that both the size of the protein and its time constant of conformational relaxation time oscillate with increasing concentration of ethanol.19 They have proposed that this oscillatory behaviour is due to the competition between the ethanol-protein, water-protein, ethanol-water, protein-protein, ethanol-ethanol and water-water interactions.19 Bagchi and co-workers have used molecular dynamics simulations to study another protein, chicken villin headpiece, in ethanol-water mixture and found that the protein unfolds partially under ambient condition.20 They have also noticed an anomalous behaviour in the unfolding pathway. While most of the reports show that proteins get denatured in the presence of aqueous mixture of ethanol,20,29,33 some of them reveal that under some conditions proteins can also get stabilized in ethanol-water mixtures.21,28 Proteins may also aggregate in the presence of aqueous mixture of alcohols.25,30,35 Nemzer et al. have reported the formation of lysozyme aggregates under acidic conditions with an ‘ethanol shock’ of 16 % (v/v) ethanol.30 Studies on such protein aggregation are important as they have found to play important roles in neurodegenerative diseases in humans including Alzheimer’s disease and Parkinson’s disease.36-38 Also, the ability to modulate the structure, and in turn functionality, of a protein by changing the alcohol concentration could prove to be helpful under many circumstances. In the present work, we have investigated the effect of ethanol-water mixture on the structure and dynamics of papain. The effect of aqueous mixture of organic solvents on papain has been previously investigated.39-41 It has been reported that papain exhibits high stability in aqueous ethanol and that the activity of papain decreases at ethanol concentrations above 60%.39 It was also found that the stability of papain could be improved by chemically modifying it.40 Even though many works related to proteins including papain in different alcohol-water mixtures could be found, the systematic study on the structural and dynamical responses are rare till now. Here, with a view of acquiring detailed structural and dynamical Page 2 of 29


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changes occurring to papain under the influence of ethanol, we have carried out steady-state fluorescence,

solvation

dynamics,

rotational

anisotropy,

fluorescence

correlation

spectroscopy and circular dichroism studies on papain in ethanol-water mixtures of different mole fractions varying from 0 to 0.6.

Materials and methods Materials Papain and guanidine hydrochloride (GnHCl) were purchased from Sigma-Aldrich and used as received. N-(7-dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA) was purchased from Molecular probes, Inc. and used without further purification. Analytical grade di-sodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Merck, India and used to prepare buffer (pH 7.4). Dialysis membrane tubing (12 kDa cut-off) was purchased from Sigma-Aldrich and used after removing the glycerol and sulfur compounds, according to the procedure given by Sigma-Aldrich. Centrifugal filter units (Amicon Ultra, 10 kDa cutoff) have been procured from Merck Millipore, Germany. HPLC grade dimethyl sulfoxide (DMSO) was purchased from S. D. Fine Chemicals Ltd. and used after distillation. Analytical grade sodium chloride and EDTA were purchased from Merck and used for the tagging reaction of Papain with DACIA. Protein Labelling and Sample Preparation The three dimensional structure of papain consists of two domains of almost equal sizes and known as L and R domains.42,43 In the cleft of these two domains cysteine-25 and histidine-159 are present.43 The free thiol group of cysteine-25 was labelled using DACIA following the procedure described by Lindahl et al.44.45 Briefly, 400 M papain solution was prepared 50 mM tris-HCl buffer (pH 7.4) containing 0.1 M NaCl and 100 M EDTA. 0.58 mg of DACIA was dissolved in 1 ml of DMSO and added drop wise to the 9 ml of papain solution with stirring under low-light conditions. The reaction mixture was stirred at 20-22 C for 2 hrs followed by dialysis against 500 mL of 1:15 (v/v) DMSO and buffer (pH 7.4, 50 mM phosphate buffer) solution at 5 °C. The dialysis medium was replaced every 12 hrs for 5 days, and then by only buffer for another 5 days. The labelled protein was concentrated using the 10 kDa cutoff centrifugal filtration unit. Samples were prepared by mixing required amount of concentrated solution of tagged papain in buffer. We have confirmed by circular dichroism spectroscopy that DACIA tagging does not altered the secondary structure of Page 3 of 29



papain. For the analysis of circular dichroism spectra, we have used CDNN software, which helps to analyse and quantify the information of far-UV CD spectra based on neural network theory as described by Böhm et al.46 Using a neural network model, it is possible to deduce the five different secondary structure fractions (𝛼-helix, parallel and antiparrel 𝛽-sheet, 𝛽-turn and random coil) of a protein molecule from its CD spectrum. The α-helicity, -sheet, -turn, random coil of untagged and DACIA tagged papain are estimated to be 24.6%, 22.1%, 16.8%, 22.1% and 23.7%, 22.6%, 17.0%, 26.7%, respectively. For AFM experiments, the samples were prepared maintaining the protein concentration around 5 µM at ethanol mole fraction XEtOH = 0.0 and XEtOH = 0.10. The sample was drop-casted on highly oriented pyrolytic graphite (HOPG). Each drop casted sample was dried in vacuum for ~12 hours before performing atomic force microscopic (AFM) investigation. All the experiments are performed at ambient conditions. Instrumentation The steady-state absorption and emission spectra were recorded in a commercial UV−visible Spectrophotometer (UV-2450, Schimadzu, Japan) and Spectrofluorimeter (FluoroMax4, JobinYvon, USA), respectively. Circular dichroism spectra were recorded in a commercial CD spectrometer (J-815, Jasco, Japan). Centrifugation was done in Eppendorf Centrifuge 5810R at 5000 rpm. Time-resolved fluorescence and anisotropy decays were collected using a commercial TCSPC setup (Life Spec II, Edinburgh Instruments, UK). Samples were excited with a 375 nm pulsed laser source (EPL-375, Edinburgh Instruments, UK). For lifetime measurements, peak counts of 8000 were collected with the emission polarizer oriented at the magic-angle polarization. Dilute ludox solution was used for measurement of instrument response function (IRF), which is found to be  120 ps. The contribution of the instrument response was nullified by deconvolution method during the fitting the fluorescence transients in Fast software, purchased from Edinburgh Instruments, UK. Femtosecond resolved fluorescence transients at different wavelengths were recorded in a commercial fluorescence up-conversion setup (FOG-100, CDP Corp., Russia). The excitation light was the second harmonic of a femtosecond Ti:Sapphire oscillator (MaiTai HP, Spectra Physics, USA) generated onto a 0.2 mm β-barium borate (BBO) crystal. The pulse width of the fundamental light (800nm) was 80 fs. The excitation light was focused on the rotating sample at magic angle polarization compared to the gate pulse passing through a delay line. The fluorescence of the sample and the gate was mixed on another 0.2 mm BBO crystal to generate the sum frequency light, which was dispersed in a monochromator and Page 4 of 29


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detected using a PMT. The power of the excitation light was kept ~3-4 mW and the overall instrument response function measured to be 250 fs. Fluorescence correlation spectroscopic (FCS) measurements were performed on an instrument assembled in our laboratory.42,47,48 Briefly, the instrument was developed on an inverted confocal microscope (Olympus IX-71, Japan). A 60X water immersion objective with NA 1.2 was used to focus the 405 nm excitation light from a CW laser source (5 mW, Optoelectronics Tech. Co. Ltd.) into the sample at a distance of 40 m away from the surface of the cover-slip. The emitted photons were collected by the same objective and focused on a multimode fiber patch chord of 25 µm diameter after passing through a dichroic mirror (ZT405rdc, Omega Optical Inc., USA) and an emission filter (FSQ-GG455, USA). Fluorescence signal was then directed towards a photon counting module (SPCM-AQRH-13FC, Excelitas) through the fiber patch chord and then to the correlator card (FLEX990EM12D, Correlator.com, USA) to generate the autocorrelation function G(). Finally, the autocorrelation curves were displayed using a LabView program routine. AFM images are acquired on a commercial atomic force microscope (Agilent-5500, USA) using high frequency (~ 280 kHz, ~ 30 N/m) PPP-NCH silicon cantilevers. All the images are post-processed and analyzed using WSxM software.49

Results Steady-state fluorescence spectroscopy study DACIA-tagged papain shows an emission maximum at 474.5 nm in its native state. In order to look at the effects of an ethanol-water mixture on papain, first we recorded the emission spectra of DACIA-tagged papain in ethanol-water mixtures of different mole fractions. The emission maximum was found to undergo a blue shift with an increase in mole fraction of ethanol. The emission maxima at different mole fractions of ethanol are given in table 1. It could be seen that at XEtOH 0.6, the emission spectra underwent a blue shift of 12 nm as compared to that in water. It is well known that papain undergoes denaturation in the presence of external agents like guanidine hydrochloride (GnHCl).50-52 To compare the effect of GnHCl on papain with that caused by ethanol-water binary mixture on the protein, we recorded the emission spectra of DACIA-tagged papain in the presence of different concentrations of GnHCl. It could be seen that in the presence of GnHCl, the emission spectrum undergoes a red shift due to the denaturation of papain as opposed to the blue shift Page 5 of 29



that occurred due to the effect of ethanol. The emission maxima of DACIA-tagged papain in the presence of different concentrations of GnHCl are also compiled in table 1. Emission spectra of DACIA-tagged papain in water, in ethanol-water mixture (XEtOH = 0.6) and in presence of 5 M GnHCl are shown in figure 1, from which the difference in effects that a binary mixture and a denaturing agent have on papain could be clearly seen.

Table 1. Emission maxima of DACIA-tagged papain in presence of different concentrations of GnHCl and in ethanol-water mixture of different mole fractions. Emission maximum [GnHCl] Emission maximum XEtOH (nm) (M) (nm) 0.00 474.5 0.00 474.5 0.05 471.5 0.50 478.0 0.10 466.5 1.00 478.5 0.20 466.0 2.00 479.0 0.30 465.0 3.00 479.5 0.40 462.5 4.00 479.5 0.60 462.5 5.00 479.5

Figure 1. Normalised emission spectra of DACIA-tagged papain (black), DACIA-tagged papain in ethanol-water mixture, XEtOH = 0.6 (blue) and DACIA-tagged papain in presence of 5 M GnHCl (red). The samples were excited at 389 nm.

Solvation dynamics study To understand more about the effects of ethanol-water mixture on the dynamics of interstitial water molecules within papain, we carried out solvation dynamics measurements of DACIA-tagged papain in water and various ethanol-water binary solvent mixtures of different mole fractions (XEtOH = 0.00, 0.05, 0.10, 0.30 and 0.50). As a representative study we are describing the results obtained pure water. The fluorescence transients at different wavelength over the steady state fluorescence spectra was measured. As for example, at 410 Page 6 of 29


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nm, the three lifetime components were found to be 0.1 ns (40%), 0.85 ns (29%) and 2.98 ns (31%) with an average lifetime of 1.21 ns and at 550 nm, the three components are 0.30 ns (14%), 1.99 ns (86%) and 3.82 ns (28%) with an average lifetime of 2.75 ns. Some of the decays are shown in figures 2a in which the rise part of fluorescence transient at a higher wavelength could be seen clearly. From the re-constructed time-resolved emission spectra the dynamic stokes shift was found to be 120 cm-1. The solvation time components were found to be 0.03 ns (17%) and 0.26 ns (83%) with average solvation time 0.22 ns.54 We have used the established procedure50 to determine the missing part of solvation dynamics and in the present case the missing part is found to be 94%. As we are missing substantial amount of solvation, drawing any conclusion only from the picosecond-nanosecond measurement may be erroneous. Hence we decided to perform the femtosecond fluorescence up-conversion study. The femtosecond fluorescent transients of some representative wavelengths are shown in figure 2b. Mukherjee et al. have explicitly described how to merge the results obtained from picosecond-nanosecond and femtosecond experiments to generate the combined C(t).50 We have followed the same process in this work too and constructed the combined TRES and C(t) (see figures 2c and 2d). We have fitted the C(t) with a tri-exponential function and determined the average solvation time from the fitting parameters. In a similar manner, solvation dynamics measurement was carried out on DACIA-tagged papain in different ethanol-water mixtures with XEtOH = 0.05, 0.10, 0.30 and 0.50. The results are tabulated in the table 2. We have also represented the representative TCSPC decays, femtosecond fluorescent transients, TRES and C(t) for XEtOH = 0.1 in figure S1 of the supporting information. It could be seen from the table that with an increase in mole fraction of ethanol the average solvation time tends to increase up to XEtOH = 0.10 and with further increase in XEtOH, the average solvation time remains almost constant. The solvent response functions of DACIA-tagged papain in different ethanol-water mixtures are also shown in figure 3, where the increment in solvation time could be seen.

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Figure 2. A few representative transient decays of DACIA-tagged papain in ethanol-water mixture of XEtOH = 0.0 collected from (a) TCSPC measurements and from (b) femtosecond up-conversion experiments. (c) Time-resolved emission spectra (TRES) constructed by combining the TCSPC and femtosecond up-conversion measurement using the fitting parameters of the transient decays and (d) the solvent response function, C(t) calculated using the peak frequencies of TRES. Table 2. Solvation time components, average solvation times, Stokes shifts and missing components of Stokes shift of DACIA-tagged papain in ethanol-water mixtures of different mole fractions. The fractional amplitudes of the solvation time components are given in the parentheses. Observed Missing 〈𝝉𝑺〉 (ps) XEtOH τ1 (ps) τ2 (ps) τ3 (ps) Stokes shift component (cm-1) (%) 0.00 2.05 (0.63) 26.1 (0.24) 298 (0.13) 46 780 61 0.05 2.02 (0.50) 26.3 (0.29) 450 (0.21) 104 980 51 0.10 2.70 (0.32) 40.2 (0.28) 514 (0.40) 218 1090 35 0.30 2.04 (0.37) 29.3 (0.24) 517 (0.39) 209 1190 27 0.50 2.33 (0.34) 34.5 (0.25) 513 (0.40) 215 1190 28

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Figure 3. Solvent response functions of DACIA-tagged papain in ethanol-water mixtures of different mole fractions.

To compare the solvation dynamics of DACIA-tagged papain in ethanol-water mixture with that in the denatured state of the protein, we carried out solvation dynamics study of the tagged papain in the presence of GnHCl. Here also we have performed both the TCSPC and femtosecond fluorescence up-conversion study and obtained the solvation times for different GnHCl concentrations, as described previo usly. The solvation times are compiled in table 3 and the solvent response functions are shown in figure 4. As can be seen, the average solvation time in the presence of GnHCl changes only slightly as compared to the increase in solvation time in ethanol-water mixtures.

Table 3. Solvation time components, average solvation times, Stokes shifts and missing components of Stokes shift of DACIA-tagged papain in the presence of different concentrations of GnHCl. Observed Missing [GnHCl] 〈𝝉𝑺〉 (ps) τ1 (ps) τ2 (ps) τ3 (ps) Stokes shift component (M) (cm-1) (%) 0 2.05 (0.63) 26.1 (0.24) 298 (0.13) 46 780 61 1 2.12 (0.60) 27.2 (0.27) 315 (0.12) 46 990 47 2 2.54 (0.52) 30.3 (0.38) 315 (0.10) 44 1040 41 3 2.41 (0.54) 29.5 (0.36) 312 (0.10) 43 1120 37 5 3.12 (0.47) 33.0 (0.46) 339 (0.07) 40 1100 36

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Figure 4. The solvent response functions of DACIA-tagged papain in the presence of different concentrations of GnHCl. The solvent response function of DACIA-tagged papain in ethanol-water mixture with XEtOH=0.1 is also shown for comparison.

Fluorescence anisotropy study Time-resolved fluorescence anisotropy study was also carried out on DACIA-tagged papain, both in the presence of GnHCl and also in ethanol-water mixtures of different mole fractions. The anisotropy decays are shown in figure 5. As could be seen from the figure, anisotropy decays are much slower in presence of ethanol as compared to the decay in water. In the presence of GnHCl (figure 5b), even though the rotational dynamics is slower than that in absence of GnHCl, the decay seems to be faster than that of DACIA-tagged papain in ethanol-water mixtures.

Figure 5. Fluorescence anisotropy decay of DACIA-tagged papain in (a) ethanol-water mixtures of different mole fractions and in (b) absence and presence of GnHCl (5 M).

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The rotational anisotropy decays are fitted using equation 1 and the rotational time constants and the residual anisotropies are tabulated in table 4. As could be seen from the table, DACIAtagged papain exhibits negligible residual anisotropy in water, while in ethanol-water mixture the residual anisotropy is much higher. In the presence of GnHCl, the residual anisotropy is higher than that of non-denatured protein, but still is lesser than in the ethanol-water mixtures. In the case of rotational time components, the value of shorter time component increases in the presence of GnHCl and in the ethanol-water mixture, while the value of longer time component decreases.

( 𝑟(𝑡) = 𝑟(∞) + [𝑟(0) ― 𝑟(∞)].[𝑎 𝑒 1

―𝑡 𝜏𝑅

) + 𝑎 𝑒(

1

―𝑡 𝜏𝑅

2

)]

2

(1)

Table 4. The components of rotational correlation times of DACIA-tagged papain in ethanol-water mixtures and in presence of 5 M GnHCl. [GnHCl] 𝝉𝑹𝟏 (ns) 𝝉𝑹𝟐 (ns) 𝝉𝑹𝟏 (ns) 𝝉𝑹𝟐 (ns) XEtOH r(∞) r(∞) (M) 0.00 0.20 (0.20) 9.50 (0.80) 0.01 0 0.20 (0.20) 9.50 (0.80) 0.01 0.05 0.63 (0.32) 7.33 (0.68) 0.11 5 0.49 (0.29) 4.28 (0.71) 0.08 0.10 0.56 (0.26) 4.90 (0.74) 0.14 0.20 0.61 (0.34) 5.91 (0.66) 0.14 0.40 0.82 (0.30) 6.78 (0.70) 0.13 0.60 0.72 (0.40) 6.89 (0.60) 0.14

Fluorescence correlation spectroscopy (FCS) study Having studied the solvation dynamics and the rotational anisotropy of DACIAtagged papain in ethanol-water mixtures and in the presence of GnHCl, we proceeded to carry out the fluorescence correlation spectroscopy measurements of the system in order to quantify the the structural changes of papain in ethanol-water mixture and GnHCl. FCS is a single molecular level technique based on the temporal fluctuation of fluorescence intensity of a probe in the observation volume. In this case, the probe, DACIA is covalently attached to the Cys-25 residue of papain. So by processing and analyzing the data, we get information about diffusion time scale (τD) and conformational fluctuation time scale (τR) of papain for which fluctuation of fluorescence intensity arises. From, τD, direct assessment of size is possible, whereas τR furnishes information about the local dynamics around the probe. Mathematically, we get these timescale after fitting the auto-correlation curves by equation 3. Here, it is to mention that, in our previous publication, we have proved that the auto-correlation traces for

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papain can not be fitted satisfactorily with single diffusion equation (equation 2) and we used equation 3 for proper fitting.42 1

(

𝑡

(

𝑡

𝐺(𝑡) = 𝑁 1 + 𝜏𝐷 1

𝐺(𝑡) = 𝑁 1 + 𝜏𝐷

―1

) (1 + ) 𝑡

―

1 2

(2)

𝜔2𝜏𝐷

―1

) (1 + ) 𝑡

𝜔2𝜏𝐷

―

1

(1 + 𝐴 ∙ exp ( ― ))

2

𝑡

𝜏𝑅

(3)

In the above equations, 𝐺(𝜏) is the autocorrelation function, N is the number of particles in the observation volume, and 𝜔 is the depth to diameter ratio of the three dimensional Gaussian volume. The diffusion coefficient (𝐷𝑡) of the molecules can be calculated from τD using equation 4 𝑟2

(4)

𝐷𝑡 = 4𝜏𝐷

In the above equation, r is the transverse radius of the observation volume. 𝜔 is calibrated by globally fitting various autocorrelation traces of rhodamine 6G (Dt of rhodamine-6G is taken as 4.14  10-6 cm2 s-1).53 And then the hydrodynamic radius is calculated using the StokesEinstein equation 𝑘𝐵𝑇

(5)

𝑟𝐻 = 6𝜋𝜂𝐷𝑡

where kB is the Boltzmann constant, T is the temperature in Kelvin and  is the viscosity of the solution. Here it is to mention that the presence of ethanol or GnHCl results in the change of viscosity and refractive index of the sample solutions, which is expected to interfere in the diffusion timescale of the protein. To nullify such changes we take two precautions.54,55 Firstly, the objective collar position are adjusted to obtain an optimized focal volume that gives the lowest τD. In this way, we nullify the effect of the change of refractive index. And secondly, the viscosity effect is canceled by doing the control experiment at every experimental point, by taking R6G (a rigid molecule; which is assumed to not undergo any structural change in the presence of ethanol or GnHCl) as the probe and then normalizing the data using equation 6. 𝑟𝑝𝑎𝑝𝑎𝑖𝑛 𝐻

=

𝑟𝑅6𝐺 𝐻

×

𝜏𝑝𝑎𝑝𝑎𝑖𝑛 𝐷

(6)

𝜏𝑅6𝐺 𝐷

In this contribution, we are mainly focusing on the structural parameter change in the presence of ethanol. Therefore, we are not concerned about the local dynamics change involved therein

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and are not reporting the τR values. The measured hydrodynamics radii of papain in presence of ethanol and GnHCl, thus obtained, are given in table 5 and the corresponding autocorrelation functions are shown in figure 6.

Table 5. Hydrodynamic radii of papain in ethanol-water mixtures of different XEtOH and in presence of different concentrations of GnHCl. Hydrodynamic radii, Hydrodynamic radii, XEtOH [GnHCl] (M) rH (Å) rH (Å) 0.00 18.4 0.0 18.4 0.05

95.4

0.5

19.3

0.10

159.8

1.0

19.7

0.20

127.9

2.0

19.7

0.30

136.5

3.0

20.7

0.40

181.7

4.0

22.8

0.50

155.2

5.0

23.3

Figure 6. Normalised autocorrelation curves and their fittings of DACIA-tagged papain in (a) ethanolwater mixtures of different XEtOH and (b) in presence of different concentrations of GnHCl. The concentration of protein was maintained at about 50 nM in all experiments.

Atomic force microscopy study To confirm the existence of papain aggregates in ethanol-water mixture, we have carried out atomic force microscopy experiments. Figure 7 shows AFM topographs of film of papain prepared with two different ethanol-water mole fractions (XEtOH = 0.00 and XEtOH = 0.10) on HOPG surface. The corresponding phase images are provided in the supporting information (figure S2). Figures 7a and 7b show AFM topographs of two different areas of papain film, deposited from XEtOH = 0.0 solution. Figure 7a shows a uniform growth of papain

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on HOPG surface. The area marked with green dashed line in figure 7b shows another area indicating a growth of papain as uniform bilayer film on HOPG surface. Figure 7c shows the typical AFM topography of papain film prepared from XEtOH = 0.10, where a distinct molecular aggregates can be visualized (marked with a few circles) and are uniformly distributed over the surface. Analysis of the size of such aggregates (using WSxM) yields an average radius of ~ 30 nm. The aggregates are observed to be inter-linked. This is most likely due to the presence of water molecules in between the aggregates. We note that even after extended pumping the morphology of the film is unaffected.

Figure 7. AFM topograph of ultra-thin film of papain on HOPG surface. (a) and (b) Sample prepared from papain in water without ethanol (XEtOH = 0.00). The area marked green in image (b) shows uniform bilayer growth on HOPG surface. (c) Sample prepared from papain in ethanol-water mixture with XEtOH = 0.10. Area marked in blue circle represents a few aggregates.

Circular dichroism spectroscopy study Circular dichroism (CD) studies help us to get information regarding the secondary structural components of a protein. We recorded the CD spectra of papain in different ethanolwater mixture and in the presence of various concentrations of GnHCl to get an idea about the changes occurring to the α-helix and β-pleated structures of papain. Some of the representative spectra are shown in figures 8a and 8b, where the difference in the changes occurring due to ethanol and GnHCl could be seen readily. Further, we have analyzed the CD data using CDNN software which helps to get quantitative information about the secondary structures.46 The data has been compiled in tables 6 and 7.

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Figure 8. Circular dichroism spectra of papain (a) in ethanol-water mixture of different mole fractions and (b) in presence of different concentrations of GnHCl. In each case, concentration of papain was 5 µM and the pathlength of cuvette was 2 mm. Table 6. Fractional components of different secondary structural elements of papain at different mole fractions of ethanol calculated using circular dichroism spectra. Fractional component (%) XEtOH α-helix β-sheet β-turn Random coil 0.00 25 21 17 37 0.05 23 24 17 36 0.10 21 26 17 36 0.20 16 31 18 35 0.40 12 37 17 34 0.60 10 41 17 32 Table 7. Fractional components of different secondary structural elements of papain in presence of different concentrations of GnHCl calculated from circular dichroism spectra. Fractional component (%)) [GnHCl] (M) α-helix β-sheet β-turn Random coil 0 25 21 17 37 1 16 26 18 40 2 14 27 17 42 3 10 30 17 43 4 10 31 17 42 5 11 30 17 42 6 10 31 17 42

Discussion The steady state emission spectra of DACIA-tagged papain in ethanol-water mixtures of different mole fractions revealed that the emission maximum of the system undergoes a monotonous blue shift with an increase in mole fraction of ethanol. The emission maximum was found to be at 474.5 nm in water, which shifts by 12 nm to 462.5 nm when Page 15 of 29



mole fraction of ethanol is 0.6. This shift in emission maximum could be due to some structural changes occurring to the protein, repositioning the fluorescent tag DACIA molecule into a more hydrophobic environment. During the denaturation of a tagged-protein, the emission maxima generally undergoes a red shift due to the action of denaturing agents as they expose the probe molecule to a less hydrophobic environment.50-52 So it would be safe to assume that the blue shift of emission maximum of DACIA-tagged papain in ethanol-water mixture is not due to the denaturation of papain. Nevertheless, in order to make sure that the change in steady state spectrum in ethanol-water mixture is not caused due to denaturation, we have looked at the change in emission maximum of DACIA-tagged papain caused due to a denaturing agent, guanidine hydrochloride (GnHCl). As expected, the emission maximum underwent a 5 nm red shift from 474.5 nm in the native state to 479.5 nm in presence of 5 M GnHCl. The variations of emission maximum in ethanol-water mixtures and that due to the action of denaturing agent are shown in figure 9, where the opposite effects caused by these two external agents could be clearly seen. In order to make sure that the blue shift in emission spectrum is not due to the change in local dielectric environment of DACIA molecules induced by ethanol, we have recorded the emission spectra of DACIA molecule in different ethanol water mixtures and the data has been compiled in table S1 of the supporting information. It could be seen that DACIA undergoes a 6.5 nm blue shift when mole fraction of ethanol is increased from 0 to 0.6 as compared to 12 nm blue shift of DACIA-tagged papain during the corresponding change in mole fraction of ethanol. We had also recorded the emission spectra of GnHCl denatured DACIA-tagged papain in different mole fractions of ethanol (see table S1 of the supporting information), which shows a shift of 7.0 nm when mole fraction is increased to 0.6. A comparison of change in emission maxima of free DACIA, DACIA-tagged papain and GnHCl (5M) denatured DACIA-tagged papain with change in mole fraction of ethanol is shown in figure S3 of the supporting information. This is to further emphasize that in presence of up to XEtOH = 0.1 the shift in emission maximum is only 0.5 nm for pure DACIA and 2.5 nm for DACIA-Papain in presence of 5M GnHCl, whereas for DACIA tagged papain the observed blue shift is 8 nm. This conclusively demonstrate that the observed blue shift of DACIA in papain in presence of ethanol is not due to the change in local dielectric environment by the solvent mixture solely, some structural rearrangement of the protein is also involved. From figure 9 it could also be noted that a major change in emission maximum occurs between XEtOH values 0 and 0.1, where a blue shift of 8 nm is observed, while further increasing XEtOH to 0.6 only causes a shift of 4 nm.

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Figure 9. Variation of emission maximum of DACIA-tagged papain with change in concentration of GnHCl and with change in mole fraction of ethanol.

Solvation time in papain increases about 2 fold in ethanol-water mixture as compared to that in water. In the case of denaturation of a protein, we expect the solvation to be either faster or to have a similar value as unfolding of the protein would make the environment of the probe molecule less confined.51 We were able to observe this effect when we studied the solvation of papain in presence of GnHCl. The presence of denaturant caused only a marginal increase in solvation time of DACIA-tagged papain as compared to the increase in solvation time observed in ethanol-water mixture. Figure 10 shows the variation of solvation time of DACIA-tagged papain in ethanol-water mixtures and in presence of GnHCl. These observations further confirm that the structural changes in papain in presence of ethanol is not the denaturation of the protein. It has been previously reported that solvation dynamics inside macromolecules becomes slower when they form aggregates58-60 This slower solvation has been attributed to the greater restriction on the mobility of solvent molecules trapped inside macromolecules when they form aggregates. In view of this, we propose that papain undergoes aggregation in aqueous mixture of ethanol, which results in a more confined environment of the probe molecule, thus resulting in slower solvation. The aggregation of the protein would also result in a more hydrophobic environment of DACIA, which results in the blue shift in emission spectrum. Rotational anisotropy study further helps to elucidate the aggregate formation of papain in ethanol-water mixtures. The shorter component of anisotropy decay corresponds to

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the rotation of DACIA molecule covalently bound to the protein, while the longer component represents the rotation of the overall protein. From table 4 it could be seen that the shorter component of rotational time is slower in ethanol-water mixtures as compared to that in water. In the ethanol-water mixtures with XEtOH 0.05 and 0.40 the shorter rotational components are 3 and 4 times slower than that in water. The aggregation of the protein in the binary mixture could be causing a restriction in the rotation of DACIA, which in turn results in slower rotational time. It could be noted that in presence of GnHCl also the shorter component becomes slower indicating that denaturation of the protein is also somehow hindering the rotation of the probe molecule.

Figure 10. Variation of the average solvation time of DACIA-tagged papain with change in concentration of GnHCl and with change in mole fraction of ethanol.

Although the values of the longer component of rotational time, which corresponds to the overall rotation of the protein decrease in ethanol-water mixtures, the amount of residual anisotropy has increased appreciably. While, in water, papain is having negligible residual anisotropy (0.01), in ethanol-water mixtures of ethanol mole fractions 0.05 and 0.60, the residual anisotropy values are 0.11 and 0.14 respectively. The higher residual anisotropy values of papain in ethanol-water mixtures could be due to the larger aggregates, which do not complete the reorientation within the excited state lifetime of the probe molecule. The higher residual anisotropy may also be the reason for the lower values of longer components of rotational times.

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The steady-state and time-resolved bulk spectroscopic measurements indicate the aggregation of papain in ethanol-water mixture and denaturation in the presence of GnHCl qualitatively. Fluorescence correlation spectroscopic study helps us to get information about the size of the moieties and thereby quantify the ehanol and GnHCl induced changes. In water, hydrodynamic radius of papain has been calculated to be 18.4 Å that matches well with previous report.42 The variation of hydrodynamic radii of papain with increasing amount of ethanol as well as with increasing concentration of GnHCl are shown in figure 11. In ethanolwater mixture, the hydrodynamic radius increases to 95.4 Å at XEtOH = 0.05 and then to 159.8 Å at XEtOH = 0.10. With further increase in the mole fraction of ethanol, the size remains almost constant. On the other hand, the size of protein moiety increases only to a small extent due to the action of GnHCl. In the presence of 5 M GnHCl, hydrodynamic radius increases to a value of 23.3 Å, which is due to the denaturation of papin.42 A huge increase in the value of the hydrodynamic radius of papain in the presence of ethanol as compared to that of the native state of papain and also to the denatured state of papain further confirms aggregate formation in the ethanol-water mixture. In many of the previous reports on protein aggregation, the aggregate formation was found to occur when the concentration of the protein is in micromolar range or higher.31,35,59,60 For our FCS experiments, we have used protein samples of concentration in the nanomolar order. The fact that we were able to detect the presence of aggregates in these samples reveal that papain can form aggregates in ethanol-water mixture even at such low concentrations. In the figure S4 of the supporting information, a variation of G(0) with increasing ethanol mole fraction and GnHCl concentration has been plotted. The increase in the value of G(0) with increasing XEtOH also suggests that the average number of molecule diffusing through the observation volume decreases, which can be correlated directly to the aggregation process. On the other hand, in presence of GnHCl, no significant change of G(0) was observed. A visual proof for the formation of aggregates of papain in ethanol-water mixtures could be obtained from AFM experiment. From figure 7, we could clearly see the presence of papain aggregates of diameter ~30 nm in ethanol-water mixture of XEtOH = 0.1, which are absent in the case of papain in pure water. Here, we want to mention that though there is a considerable difference in the hydrodynamic radius of papain aggregates obtained through FCS and AFM measurements, the formation of aggregating species is unambiguously proved. The difference in the size of papain aggregates may be attributed to the fact that while in FCS the size is measured in the solution phase, in case of AFM study the measurements are

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conducted on the aggregated proteins deposited on a solid surface. The aggregates would be flattened to some extend when they come in contact with the surface, which leads to an increase in its diameter.

Figure 11. Hydrodynamic radii of papain in ethanol-water mixtures of different XEtOH and in presence of different concentrations of GnHCl.

Circular dichroism measurements can help us to get a clearer picture about the secondary structure of proteins and the changes induced on it due to the changes in its environment. Analysis of CD spectra using CDNN software can deliver us information about the fractional components of different secondary structural elements including α-helix, βsheet and β-turn and the fraction of random coil in the protein moiety. It has been previously reported in literature that, proteins like human serum albumin and bovine serum albumin aggregates by the formation of β-sheet.61-63 Also, in most of the cases the aggregates of proteins are found to be rich in β-sheet. We have monitored the change in the fractional components of different secondary elements of papain with an increasing mole fraction of ethanol (figure 12a). It could be seen that the β-sheet content increases from 21% in water to 41% in ethanol-water mixture with XEtOH=0.60 at the expense of α-helix (25% to 10%) and random coil (37% to 32%) content. From this analysis, we propose that the enrichment of the β-sheet content in presence of ethanol may be the reason for the aggregate formation. Similar analysis for papain in presence of GnHCl (figure 12b) showed a decrease in the amount αhelix structures and an increase in the amount of random coil, which is expected during denaturation. An increase in the amount of β-sheet is also observed during the denaturation process (from 37% to 42%), even though this increase is smaller than the corresponding

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change occurred in the case of papain in ethanol-water mixtures while the amount of β-turn remains almost constant in this case as well.

Figure 12. Variation in the amount of different secondary structural components of papain with (a) change in mole fraction of ethanol and (b) with change in concentration of GnHCl.

Conclusion In this report we have scrutinized the effect of ethanol-water binary solvent mixture on the structural response of papain by various bulk and single molecular level spectroscopic measurement and compared the result with GnHCl induced structural change of papain. From the spectroscopic studies, we can conclude that GnHCl denaturates papain, whereas ethanol induces its aggregation. A strong corelation between structural change at single molecular level measurement and secondary structural change from the circular dichroism measurement has been found. Chemical denaturation of papain by GnHCl is associated with the loss of helicity with a concomitant increase of random-coil and a small increase of -sheet. Whereas, ethanol induced aggregation of papain is associated with the formation of high prercentage of

-sheet. The findings are supported by steady-state experiment, solvation dynamics and the rotational anisotropy of the protein in ethanol-water mixture and in GnHCl. The visual conformation of aggregation formation of papain in the presence of ethanol comes from the atomic force microscopy experiments. Our result suggests that two chemical constituent can interact with a protein in a very different way, reminding us of the tremandous degree of complexity involved in the biological system. Another surprising highlight of the report is that papain was found to form aggregates in ethanol-water mixture at concentrations as low as 50 nM. As a whole, the global and local structural change of papain in ethanol-water binary

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solvent mixture and also in presence GnHCl has been summarised, quantified and explained. Papain, being tremendously important in industry, are required to be protected against aggregation. In this paper, we showed that papain is very much prone to aggregation in the presence of ethanol. In our upcoming projects, we will come up with some strategy to prevent this aggregation.

Acknowledgment VM thanks Indian Institute of Technology Kanpur for fellowship. ND and VM acknowledges Council of Scientific and Industrial Research (CSIR, Government of India) for providing fellowship. AD acknowledges Ministry of Electronics and Information Technology, Govt. of India for scholarship. Authors thank Dr. T. G. Gopakumar, Department of Chemistry, IIT Kanpur for fruitful discussion on AFM. This work is financially supported by Science and Engineering Research Board, Government of India (Grant No. EMR/2016/006555) and IIT Kanpur.

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A Spectroscopic Insight on Ethanol Induced Aggregation of Papain

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