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water binary mixture probed by terahertz spectroscopy
Biophysical Chemistry 216 (2016) 31–36
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Preferential solvation of lysozyme in dimethyl sulfoxide/water binary mixture probed by terahertz spectroscopy Dipak Kumar Das 1, Animesh Patra 1, Rajib Kumar Mitra ⁎ Department of Chemical, Biological and Macromolecular Sciences, S.N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700098, India
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Addition of DMSO in lysozyme aqueous solution induces noticeable conformational changes. • DMSO gets preferentially absorbed at the protein surface leading to the conformational changes. • Collective hydration dynamics at the protein surface follows the trace of DMSO induced conformational changes.
a r t i c l e
i n f o
Article history: Received 25 February 2016 Received in revised form 14 June 2016 Accepted 14 June 2016 Available online 25 June 2016 Keywords: Preferential solvation Collective hydration Terahertz spectroscopy Lysozyme Dimethyl sulfoxide
a b s t r a c t We report the changes in the hydration dynamics around a model protein hen egg white lysozyme (HEWL) in water–dimethyl sulfoxide (DMSO) binary mixture using THz time domain spectroscopy (TTDS) technique. DMSO molecules get preferentially solvated at the protein surface, as indicated by circular dichroism (CD) and Fourier transform infrared (FTIR) study in the mid-infrared region, resulting in a conformational change in the protein, which consequently modifies the associated hydration dynamics. As a control we also study the collective hydration dynamics of water–DMSO binary mixture and it is found that it follows a non-ideal behavior owing to the formation of DMSO–water clusters. It is observed that the cooperative dynamics of water at the protein surface does follow the DMSO-mediated conformational modulation of the protein. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Aqueous environment of biomolecules often plays the key roles in both inter and intra-molecular interactions which are responsible for ⁎ Corresponding author. E-mail addresses: [email protected] (D.K. Das), [email protected] (R.K. Mitra). 1 Contributed equally.
http://dx.doi.org/10.1016/j.bpc.2016.06.002 0301-4622/© 2016 Elsevier B.V. All rights reserved.
their stabilization; such interactions include hydrogen bonding, hydrophobic interactions, ionic bonding etc. [1,2] Addition of otherwise indifferent components like sugar, salts, organic solvents etc. can influence protein conformation [3] either by direct interaction or through preferential solvation, in which the additive preferentially or selectively exists in the protein solvation shell [4,5]. Owing to this unique ability binary solvents can control as well as tune the structure of protein or bio-molecules, and thus have received considerable attention in recent years.
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Water–dimethyl sulfoxide (DMSO) binary mixture has been found to be of potential interest as it plays a significant role in the field of chemistry, physics, biology and pharmacology [6–10]. Owing to the specific hydrogen bonding ability of DMSO the binary mixture often exhibits some unique physicochemical behavior [8]. DMSO acts as a protein stabilizer at low concentration whereas it destabilized proteins at high concentration which makes this binary mixture to be used as a stabilizer, a denaturant, an activator, or an inhibitor [11–14]. The effect of this binary mixture on the conformational stability of a model protein lysozyme has previously been studied by several research groups. Far-UV CD measurements concluded a native to unfolded structural transition of lysozyme at low DMSO concentration (XDMSO ~ 0.06) [15]. At higher DMSO concentration the protein undergoes broad structural transition. NMR relaxation study by Johannesson et al. [14] has established a direct interaction of DMSO molecules with the active cleft of lysozyme. Kamiyama et al. [16] made a detailed thermodynamic study to establish a preferential solvation of lysozyme by DMSO in the binary mixture. Kovrigin et al. [17] have carried out a comparative study on the preferential solvation of DMSO and four other organic solvents during thermal denaturation of lysozyme and concluded that the initial interaction of the exposed groups of the denatured protein and organic solvent is non-specific in nature. Voets et al. [11] used small angle neutron scattering (SANS) technique to identify unfolded to partially collapsed denatured state of lysozyme with increasing DMSO concentration. A recent Molecular Dynamics (MD) simulation study by Roy et al. [18] has concluded the protein to pass through several conformational states as the composition of the mixture changes. Fluorescence correlation spectroscopy study by Ghosh et al. [19] by labeling lysozyme with Alexa 488 concluded the hydrodynamic radius of the protein to undergo systematic changes in the low DMSO concentration region (b30 mol%). All these above mentioned studies have unambiguously concluded substantial effect of DMSO on the protein conformation, however, less attention has been paid on the dynamics of the hydration layer associated with the protein as it undergoes conformational changes. In this contribution we probe the hydration dynamics of hen egg white lysozyme (HEWL) at different compositions of the DMSO–water binary mixture, mainly in the range of 0–0.5 mol fraction of DMSO, using THz time domain spectroscopy (TTDS) technique. THz spectroscopy has recently emerged as a potential tool to label free determination of the collective hydrogen bond dynamics which extends up to several hydration layers [20] around solutes [21,22], also applicable for measuring the collective hydration dynamics at various compositions of binary mixtures [23] and biomolecules [24,25]. This technique offers a unique advantage to examine the fate of the H-bonded network dynamics of water in otherwise less polar environments including biological interfaces [26,27]. Various frequency-dependent optical parameters of the solution (viz. absorption coefficient, α(ν), complex refractive index, n ðνÞ, complex dielectric constants, ε(ν)) etc. can be extracted from a single measurement [28]. In this frequency region it is possible to locate the low frequency collective modes that are responsible for the direct flow of conformational energy (as a result of the changes of interaction pattern with varying the concentration of protein in buffer medium) in many biological processes [25]. There have been a few earlier studies on the collective dynamics in partially or fully hydrated lysozyme using THz spectroscopy [29–31], however, a detailed understanding on the changes in the hydration dynamics in its different conformational states has not yet been attempted. We determine the changes in the tertiary structure of the protein in presence of DMSO using CD spectroscopy in the near-UV region. We obtain the hydrogen bonded structure of water by measuring the O-H bond stretch in the mid-IR region using FTIR technique. We investigate the hydration dynamics around HEWL at different compositions of the binary mixture using TTDS. The primary focus of this study is to understand whether the perturbation experienced by the protein in DMSO–water mixture has its imprint on the associated hydration structure of the protein
and how the preferential solvation of protein by DMSO associates the altered dynamics. 2. Materials and methods Hen egg white lysozyme (HEWL, crystallized lyophilized powder) and dimethyl sulfoxide (DMSO, HPLC grade) were procured from Sigma-Aldrich and were used as received. 10 mg ml−1 aqueous-HEWL solution was prepared in Milli-Q water. This low concentration of HEWL was used in order to avoid any clustering [32]. Milli-Q water was used for all the binary mixture preparation. In all the measurements only freshly prepared aqueous-HEWL–DMSO solutions were used. FTIR spectra of water-DMSO and aqueous-HEWL-DMSO solutions were measured using FTIR 6300 JASCO spectrometer. The O-H stretch of HOD was measured in a solution of 4% D2O in water. A 15 μm spacer was used for all the FTIR measurements. Circular dichroism (CD) measurements in the near-UV region were performed in a JASCO J-815 spectrometer using 1 mm cuvette. Due to the strong absorption of DMSO in the ~250 nm wavelength region, we do not perform the CD experiments in the far-UV region. The sample scan speed was kept at 50 nm min−1 with response time of 2 s. Three CD spectra were recorded in continuous mode and averaged for each CD experiment. THz-time domain spectroscopy (TTDS) measurements were carried out in a commercial THz spectrophotometer (TERA K8, Menlo Systems) [33]. In brief, a 780 nm Er doped fiber laser (b 100 fs pulse width (FWHM), 100 MHz repetition rate) is split into pump and probe beams of equal power (~10 mW) using a polarizing beam splitter. The pump beam excites the THz emitter antenna producing a THz radiation having a bandwidth of ~ 3.0 THz (N 60 dB). This THz radiation after transmitting through the sample is focused on a THz detector antenna which is gated by the probe laser beam. The THz antennas are gold dipoles with a dipole gap of 5 μm deposited on LTG–Ga As substrate. To avoid water vapor absorption, all the measurements were carried out in dry nitrogen atmosphere with a controlled humidity of b10% in a liquid cell (Bruker, model A-145) using z-cut quartz windows and Teflon spacer of 100 μm thickness. The samples were reloaded for five times in the sample cell and nine full scans were averaged together to minimize the error in the results. By varying the time delay between the probe and the pump beam the amplitude and phase of the THz electric field were measured as a function of time. The frequency dependent power and phase of the transmitted pulse is obtained using Fourier analysis of the measured electric field amplitude ETHz(t). TTDS involves coherent detection mechanism and can thus measure both the amplitude and the phase of radiation in a single measurement and can provide with information on frequency dependent optical parameters of the system. Subsequently, the frequency dependent absorption coefficient α(ν) (power attenuation) and index of refraction n(ν) (delay of the THz pulse) can be obtained. 3. Results and discussions 3.1. Circular dichroism (CD) studies Fig. 1 represents the effect of DMSO on the protein's tertiary structure as extracted from the near-UV CD spectra. The peaks observed at ~287 and 292 nm (Fig. 1a) are due to the transitions of the tryptophan residues of lysozyme [34] and thus correspond to the tertiary structure of the protein. The intensity of the CD spectrum initially increases with increasing XDMSO to reach a maximum at XDMSO ~ 0.25 beyond which it decreases. Fig. 1b shows the DMSO induced variation in the CD signal intensity at 287 and 292 nm. In the low DMSO concentration region the spectral pattern remains almost unchanged where DMSO acts as a protein stabilizer. Previously reported fluorescence studies of the intrinsic tryptophan also concluded a marked blue shift of the emission maximum pointing towards a stabilization of the protein [16,19]. Earlier studies have confirmed that the changes in the CD signals vis-à-vis the
D.K. Das et al. / Biophysical Chemistry 216 (2016) 31–36
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Fig. 1. (a) CD spectra of aqueous-HEWL solution at different DMSO concentrations in the near-UV region. (b) CD signal at 292 nm and 287 nm as a function of XDMSO. The dotted lines are guide to the eyes.
protein conformation is not due to any change in the pH of the binary mixture [17,19] and thus can only be attributed to the specific DMSO– water interactions. A recent simulation study has reported that some of the exposed hydrophobic residues including tryptophan, that usually reside near the active site of lysozyme, get affected by the presence of DMSO up to XDMSO ~ 0.2 [18]. The present CD measurements corroborate this simulation results. On further increase in the DMSO concentration the protein conformation changes as evidenced from the change in the spectral pattern coupled with a sharp drop in the intensity (Fig. 1b). CD study thus establishes conformational changes in the protein structure due to its specific interaction with DMSO. 3.2. FTIR studies As a control, we measure the FTIR spectra of the O-H stretching mode in water–DMSO binary mixtures (Fig. S1, Supporting information section). It is found that the intensity of the low frequency mode decreases with increasing DMSO concentration keeping the OH peak position unaltered. In addition, the full width at half maxima (FWHM) of the spectrum gets narrower, especially in the high DMSO concentration region. We deconvolute the O-H spectra into three Gaussian curves to quantify the relative contributions of different types of H-bonded water molecules: strongly H-bonded water or HW (appear in the lower frequency region), distorted water (DW) and isolated water (IW) (higher frequency region). HW is generally assigned to the water molecules having H-bonded coordination number close to four. Such a component closely relates the OH band observed in ice and this type of water is also denoted as the network water [35]. DW is defined as the water molecules which have the average degree of connections larger than that of the dimers or trimers but lower than those participating to the tetrahedral water networks. IW is defined as those water molecules which are poorly connected to their environments; mainly these are interfacial monomer waters or the water molecules which do not produce strong hydrogen bonds with neighboring water molecules [36]. Two representative deconvoluted spectra at XDMSO = 0 and XDMSO = 0.45 are shown in Fig. S2, (Supporting information section). The fit parameter for the deconvolution of HW, DW and IW are shown in Table S1. The O-H stretching spectrum of aqueous-HEWL solution is found to be comparable to that of pure water with only marginal changes in the HW and DW populations (Fig. S3, Supporting information section). However, as DMSO is added into the protein solution there occurs noticeable change in the absorption profile. A representative deconvolution at XDMSO = 0.45 is shown in Fig. S4 (Supporting information section). The fit parameter for the deconvolution of HW, DW and IW are also shown in Table S2. The width of the IW increases slightly with increasing DMSO concentration both in presence and in absence of the protein. It should be mentioned here that as IW molecules are poorly connected to their environments, they might not be fully isolated. However, it is also to be noted that the relative abundance of such molecules is very low and also does not change noticeably with
composition (Tables S1 and S2). So a reasonable discussion could be made on the basis of changes in HW and DW molecules only. Comparing Figs. S2 and S4, a striking dissimilarity is envisaged. We plot the relative abundance of HW and DW in absence and in presence of the protein (Fig. 2a,b). It can be noted here that the relative population of IW is very small and does not undergo noticeable change with DMSO. The HW and DW profile shows distinctly opposite trends in presence of the protein (Fig. 2). For the water–DMSO mixture the HW population decreases gradually with a concomitant increase in the DW population (the blue square symbols, Fig. 2). At low DMSO concentration (XDMSO b 0.1), the DMSO molecules tend to form micro-micellar aggregates involving 3–4 methyl moieties [10] which leads to a marginal change in the relative abundances. On further addition of DMSO, the micelle-like structures break up to produce extended structures involving extensive hydrogen bonding with water molecules. This in turn produces the observed sharp increase in the DW abundance at the expense of HW (Fig. 2). To explain the trends in the water–DMSO–HEWL ternary system one needs to consider the various interactions present: water–water, water–DMSO, water–protein as well as the protein–DMSO interactions. In water-HEWL binary solution the result can be described by considering the water–water and water–protein interactions only. As DMSO is introduced into the protein solution, it preferentially solvates the protein surface [16] expelling a fraction of the protein solvating water molecules into the bulk. This increases the relative abundance of water–water H-bond at the expense of water–DMSO H-bonds. The otherwise observed increase in the DW abundance in the binary mixture eventually reduces to a decrease in the DW abundance in presence of the protein (Fig. 2). Interaction of DMSO with HEWL changes the conformational flexibility as well as the solvent accessible surface area (SASA) of the protein [10]. One thus also has to additionally consider the protein–DMSO interaction which forms at the cost of water–DMSO interaction resulting in an overall decrease in the DW content. Beyond X DMSO ~ 0.3, the added DMSO molecules start interacting mostly with water and the DW abundance increases slightly. The FTIR study thus affirms the change in the hydration structure around HEWL as a result of the preferential solvation by DMSO. 3.3. TTDS study The THz absorption spectra for water–DMSO binary mixtures are shown in Fig. 3a. It can be noted here that absorption coefficient α(ν) in the THz frequency region is correlated with the collective dynamics of water and therefore is a useful parameter to study [20,27]. THz absorption decreases with increasing DMSO content; a closer insight reveals that the decrease is not linear. In order to obtain a more quantitative picture we plot α measured at 1 THz as a function of DMSO concentration (Fig. 3b). It is observed that α does not follow any linear trend with X DMSO , rather it passes through a minimum
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Fig. 2. Relative population of (a) hydrogen bonded water and (b) distorted water following the deconvolution (Gaussian) of MIR-FTIR spectra in water/DMSO/HEWL solutions at different DMSO concentrations. The broken lines are guide to the eyes.
indicating a non-ideal hydration behavior in the mixture. For an ideal mixture, the absorption coefficient (α id) obeys a two component model: α id ¼ α w φw þ α D φD
ð1Þ
where φ denotes the volume fraction. We plot αid in the same Fig. 3b (red dotted line) for comparison and observe marked deviation from ideality. To estimate the extent of deviation we plot Δα/αid (where Δα = α0-αid) as a function of XDMSO in the inset and observe a minimum at XDMSO ~ 0.25. It, therefore, is evident that a simple two component model is insufficient to account for the observed changes in α. Eq. (1) thus needs the introduction of a new term which takes care of the DMSO–water complex formation, α 0 ¼ α w φw þ α D φD þ α w−D φw−D
ð2Þ
where the last term appears due to the formation of water–DMSO complex structure. It is important to note that the dynamic hydration shell around solute molecules, which involves the ultrafast rearrangement in the associated hydrogen-bonded network, offers an absorption coefficient different from that of pure water [37]. The negative deviation from the ideality in α (Fig. 3b) can emanate from the term αw-D , or from a negative volume of mixing [38] or both. The negative excess molar volume of mixing (VEm) in DMSOwater mixture is indeed one of the responsible terms that contributes to the negative deviation in α. However, VEm has been reported to pass through a minimum at a relatively higher XDMSO ~ 0.4 [38], which, therefore indicates that hydration dynamics of the clustered structure in the mixture (as manifested by αw-D) does also contribute to the observed non-ideality.
With the background of anomalous water–DMSO interaction we now investigate the preferential solvation of the protein by DMSO. We measure the α(ν) profile of HEWL in DMSO–water solutions at different XDMSO. Some representative profiles are shown in Fig. 4a. In pure aqueous solution (uppermost panel) the absorption profile of the protein solution lies below that of pure water, which is explainable from the fact that water has high THz absorption than that of the proteins. However, a simple two component consideration is again insufficient to account for the observed change and for a more realistic rationale one should also consider the hydration sheathe of the protein, which offers different absorption coefficient than that of pure water [25,39]. The αobs(ν) thus cumulates contributions from bulk water, hydration water and protein [39]. It is now interesting to study how this hydration dynamics changes as DMSO induces the conformational changes. As discussed earlier, addition of DMSO invokes some additional pairwise interactions like water–DMSO, protein–DMSO and DMSO–DMSO. To compare the hydration structure of the protein we compare the absorption profile of the DMSO–water mixture at different compositions in absence and in presence of the protein. Some representative profiles are shows in the middle and lower panels of Fig. 4a. It is evident that the profiles change as XDMSO increases. At XDMSO = 0.05, the protein hydration curve lies well below the binary solution curve whereas at XDMSO = 0.25, it lies slightly above. In order to intrude into a more quantitative insight we plot the relative change in the absorption coefficient at 1 THz, denoted by Δα/α0 (where α0 is the absorption coefficient of the binary solution measured at 1 THz and Δα = αp-α0, where αp is the absorption coefficient measured at 1 THz in presence of the protein). The profile shows a minimum at XDMSO = 0.05 and then increases up to XDMSO = 0.15 beyond which it slowly decreases (Fig. 4b). The observation can be explained on the basis of the hydration around the protein as DMSO competes with water as a solvent. As DMSO is added into the protein solution, it replaces the hydrated
Fig. 3. (a) Frequency dependent Absorption coefficient α of water–DMSO binary mixture at different concentrations. The arrow indicates increasing DMSO concentration. (b) α measured at 1 THz for DMSO–water binary mixture as a function of DMSO concentration. The solid line is a guide to the eyes. The broken red line represents the ideal values of α. The inset shows the relative change in α.
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Fig. 4. (a) Absorption coefficient of water–DMSO mixtures in absence (blue, solid line) and in presence (red broken line) of HEWL. (b) Relative change in α of aqueous HEWL/DMSO solution as a function of DMSO concentration.
water (around protein) into the bulk and as a result the relative abundance of hydrated and bulk water changes. The preferential solvation of DMSO also in its turn disrupts the water-DMSO cluster structures. This effect is manifested in the changes in Δα. At XDMSO = 0.05, Δα decreases sharply and such a considerable change at a low DMSO content is intriguing. The major rationale behind this change perhaps emanates from the fact that the protein size increases by two folds at this DMSO concentration as evidenced by Ghosh et al. using FCS studies [19]. This in turn increases the effective volume fraction of the low absorbing protein molecules and consequently the overall Δα value decreases rapidly. As the concentration of DMSO increases further, the protein starts rebuilding its tertiary structure and the hydrodynamic radius approaches its native value [19]. During the process of refolding [18] the nonpolar solvent accessible surface area (SASA) of the protein increases leading to preferential accumulation of more DMSO molecules at the protein surface [16]. As a result of this preferential solvation the DMSO–water complex, otherwise present in the neat binary mixture, starts melting giving rise to more water–water hydrogen bonded network (compare DW and HW in Fig. 2a,b). The change in the relative volume fraction in presence and in absence of the protein induces an overall increase in the Δα at XDMSO = 0.1 compared to that in XDMSO = 0.05. Intriguingly, at XDMSO = 0.15, Δα has a positive value. The positive value of Δα at this composition can be rationalized from a relatively high positive contribution from the absorption of the protein hydration layer which overwhelms the otherwise negative contribution due to the preferential solvation of DMSO. It is important to note that at this composition the nonpolar SASA of the protein reaches its maximum [18], this in turn can change absorption due to the protein hydration layer and make the overall Δα positive. Further increase in the DMSO concentration decreases the nonpolar SASA [18], and also saturates the melting of the DMSO–water complex (see Fig. 2), which makes the Δα to decrease (Fig. 4b). 4. Conclusions Our study is aimed to understand whether the DMSO induced conformation changes in lysozyme conformation perturbs its hydration dynamics. CD study establishes a marked change in the protein tertiary structure in presence of DMSO. Preferential solvation of DMSO at the HEWL surface, as established from the MIR FTIR analysis, results in a relative decrease of DW population at the expense of HW content. The hydration dynamics at the protein surface is obtained from the relative change in the THz absorption coefficient (Δα/α0) which shows a negative minimum at XDMSO = 0.05 and a positive value at XDMSO = 0.15. The results are explained on the basis of the relative contributions of the absorption due to the various components present in the system.
The observed minimum is found to be due to the increased size of the protein while the positive value is attributed to the increased SASA and consequent increased hydration of the protein surface. Our study affirms that the collective hydration dynamics at the protein surface does follow the conformational changes in the protein caused by the preferential solvation of DMSO.
Acknowledgements RKM acknowledges DST, India for a research grant (SB/S1/PC-056/ 2013).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bpc.2016.06.002.
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Preferential solvation of lysozyme in dimethyl sulfoxide/water binary mixture probed by terahertz spectroscopy Dipak Kumar Das 1, Animesh Patra 1, Rajib Kumar Mitra ⁎ Department of Chemical, Biological and Macromolecular Sciences, S.N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700098, India
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Addition of DMSO in lysozyme aqueous solution induces noticeable conformational changes. • DMSO gets preferentially absorbed at the protein surface leading to the conformational changes. • Collective hydration dynamics at the protein surface follows the trace of DMSO induced conformational changes.
a r t i c l e
i n f o
Article history: Received 25 February 2016 Received in revised form 14 June 2016 Accepted 14 June 2016 Available online 25 June 2016 Keywords: Preferential solvation Collective hydration Terahertz spectroscopy Lysozyme Dimethyl sulfoxide
a b s t r a c t We report the changes in the hydration dynamics around a model protein hen egg white lysozyme (HEWL) in water–dimethyl sulfoxide (DMSO) binary mixture using THz time domain spectroscopy (TTDS) technique. DMSO molecules get preferentially solvated at the protein surface, as indicated by circular dichroism (CD) and Fourier transform infrared (FTIR) study in the mid-infrared region, resulting in a conformational change in the protein, which consequently modifies the associated hydration dynamics. As a control we also study the collective hydration dynamics of water–DMSO binary mixture and it is found that it follows a non-ideal behavior owing to the formation of DMSO–water clusters. It is observed that the cooperative dynamics of water at the protein surface does follow the DMSO-mediated conformational modulation of the protein. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Aqueous environment of biomolecules often plays the key roles in both inter and intra-molecular interactions which are responsible for ⁎ Corresponding author. E-mail addresses: [email protected] (D.K. Das), [email protected] (R.K. Mitra). 1 Contributed equally.
http://dx.doi.org/10.1016/j.bpc.2016.06.002 0301-4622/© 2016 Elsevier B.V. All rights reserved.
their stabilization; such interactions include hydrogen bonding, hydrophobic interactions, ionic bonding etc. [1,2] Addition of otherwise indifferent components like sugar, salts, organic solvents etc. can influence protein conformation [3] either by direct interaction or through preferential solvation, in which the additive preferentially or selectively exists in the protein solvation shell [4,5]. Owing to this unique ability binary solvents can control as well as tune the structure of protein or bio-molecules, and thus have received considerable attention in recent years.
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Water–dimethyl sulfoxide (DMSO) binary mixture has been found to be of potential interest as it plays a significant role in the field of chemistry, physics, biology and pharmacology [6–10]. Owing to the specific hydrogen bonding ability of DMSO the binary mixture often exhibits some unique physicochemical behavior [8]. DMSO acts as a protein stabilizer at low concentration whereas it destabilized proteins at high concentration which makes this binary mixture to be used as a stabilizer, a denaturant, an activator, or an inhibitor [11–14]. The effect of this binary mixture on the conformational stability of a model protein lysozyme has previously been studied by several research groups. Far-UV CD measurements concluded a native to unfolded structural transition of lysozyme at low DMSO concentration (XDMSO ~ 0.06) [15]. At higher DMSO concentration the protein undergoes broad structural transition. NMR relaxation study by Johannesson et al. [14] has established a direct interaction of DMSO molecules with the active cleft of lysozyme. Kamiyama et al. [16] made a detailed thermodynamic study to establish a preferential solvation of lysozyme by DMSO in the binary mixture. Kovrigin et al. [17] have carried out a comparative study on the preferential solvation of DMSO and four other organic solvents during thermal denaturation of lysozyme and concluded that the initial interaction of the exposed groups of the denatured protein and organic solvent is non-specific in nature. Voets et al. [11] used small angle neutron scattering (SANS) technique to identify unfolded to partially collapsed denatured state of lysozyme with increasing DMSO concentration. A recent Molecular Dynamics (MD) simulation study by Roy et al. [18] has concluded the protein to pass through several conformational states as the composition of the mixture changes. Fluorescence correlation spectroscopy study by Ghosh et al. [19] by labeling lysozyme with Alexa 488 concluded the hydrodynamic radius of the protein to undergo systematic changes in the low DMSO concentration region (b30 mol%). All these above mentioned studies have unambiguously concluded substantial effect of DMSO on the protein conformation, however, less attention has been paid on the dynamics of the hydration layer associated with the protein as it undergoes conformational changes. In this contribution we probe the hydration dynamics of hen egg white lysozyme (HEWL) at different compositions of the DMSO–water binary mixture, mainly in the range of 0–0.5 mol fraction of DMSO, using THz time domain spectroscopy (TTDS) technique. THz spectroscopy has recently emerged as a potential tool to label free determination of the collective hydrogen bond dynamics which extends up to several hydration layers [20] around solutes [21,22], also applicable for measuring the collective hydration dynamics at various compositions of binary mixtures [23] and biomolecules [24,25]. This technique offers a unique advantage to examine the fate of the H-bonded network dynamics of water in otherwise less polar environments including biological interfaces [26,27]. Various frequency-dependent optical parameters of the solution (viz. absorption coefficient, α(ν), complex refractive index, n ðνÞ, complex dielectric constants, ε(ν)) etc. can be extracted from a single measurement [28]. In this frequency region it is possible to locate the low frequency collective modes that are responsible for the direct flow of conformational energy (as a result of the changes of interaction pattern with varying the concentration of protein in buffer medium) in many biological processes [25]. There have been a few earlier studies on the collective dynamics in partially or fully hydrated lysozyme using THz spectroscopy [29–31], however, a detailed understanding on the changes in the hydration dynamics in its different conformational states has not yet been attempted. We determine the changes in the tertiary structure of the protein in presence of DMSO using CD spectroscopy in the near-UV region. We obtain the hydrogen bonded structure of water by measuring the O-H bond stretch in the mid-IR region using FTIR technique. We investigate the hydration dynamics around HEWL at different compositions of the binary mixture using TTDS. The primary focus of this study is to understand whether the perturbation experienced by the protein in DMSO–water mixture has its imprint on the associated hydration structure of the protein
and how the preferential solvation of protein by DMSO associates the altered dynamics. 2. Materials and methods Hen egg white lysozyme (HEWL, crystallized lyophilized powder) and dimethyl sulfoxide (DMSO, HPLC grade) were procured from Sigma-Aldrich and were used as received. 10 mg ml−1 aqueous-HEWL solution was prepared in Milli-Q water. This low concentration of HEWL was used in order to avoid any clustering [32]. Milli-Q water was used for all the binary mixture preparation. In all the measurements only freshly prepared aqueous-HEWL–DMSO solutions were used. FTIR spectra of water-DMSO and aqueous-HEWL-DMSO solutions were measured using FTIR 6300 JASCO spectrometer. The O-H stretch of HOD was measured in a solution of 4% D2O in water. A 15 μm spacer was used for all the FTIR measurements. Circular dichroism (CD) measurements in the near-UV region were performed in a JASCO J-815 spectrometer using 1 mm cuvette. Due to the strong absorption of DMSO in the ~250 nm wavelength region, we do not perform the CD experiments in the far-UV region. The sample scan speed was kept at 50 nm min−1 with response time of 2 s. Three CD spectra were recorded in continuous mode and averaged for each CD experiment. THz-time domain spectroscopy (TTDS) measurements were carried out in a commercial THz spectrophotometer (TERA K8, Menlo Systems) [33]. In brief, a 780 nm Er doped fiber laser (b 100 fs pulse width (FWHM), 100 MHz repetition rate) is split into pump and probe beams of equal power (~10 mW) using a polarizing beam splitter. The pump beam excites the THz emitter antenna producing a THz radiation having a bandwidth of ~ 3.0 THz (N 60 dB). This THz radiation after transmitting through the sample is focused on a THz detector antenna which is gated by the probe laser beam. The THz antennas are gold dipoles with a dipole gap of 5 μm deposited on LTG–Ga As substrate. To avoid water vapor absorption, all the measurements were carried out in dry nitrogen atmosphere with a controlled humidity of b10% in a liquid cell (Bruker, model A-145) using z-cut quartz windows and Teflon spacer of 100 μm thickness. The samples were reloaded for five times in the sample cell and nine full scans were averaged together to minimize the error in the results. By varying the time delay between the probe and the pump beam the amplitude and phase of the THz electric field were measured as a function of time. The frequency dependent power and phase of the transmitted pulse is obtained using Fourier analysis of the measured electric field amplitude ETHz(t). TTDS involves coherent detection mechanism and can thus measure both the amplitude and the phase of radiation in a single measurement and can provide with information on frequency dependent optical parameters of the system. Subsequently, the frequency dependent absorption coefficient α(ν) (power attenuation) and index of refraction n(ν) (delay of the THz pulse) can be obtained. 3. Results and discussions 3.1. Circular dichroism (CD) studies Fig. 1 represents the effect of DMSO on the protein's tertiary structure as extracted from the near-UV CD spectra. The peaks observed at ~287 and 292 nm (Fig. 1a) are due to the transitions of the tryptophan residues of lysozyme [34] and thus correspond to the tertiary structure of the protein. The intensity of the CD spectrum initially increases with increasing XDMSO to reach a maximum at XDMSO ~ 0.25 beyond which it decreases. Fig. 1b shows the DMSO induced variation in the CD signal intensity at 287 and 292 nm. In the low DMSO concentration region the spectral pattern remains almost unchanged where DMSO acts as a protein stabilizer. Previously reported fluorescence studies of the intrinsic tryptophan also concluded a marked blue shift of the emission maximum pointing towards a stabilization of the protein [16,19]. Earlier studies have confirmed that the changes in the CD signals vis-à-vis the
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Fig. 1. (a) CD spectra of aqueous-HEWL solution at different DMSO concentrations in the near-UV region. (b) CD signal at 292 nm and 287 nm as a function of XDMSO. The dotted lines are guide to the eyes.
protein conformation is not due to any change in the pH of the binary mixture [17,19] and thus can only be attributed to the specific DMSO– water interactions. A recent simulation study has reported that some of the exposed hydrophobic residues including tryptophan, that usually reside near the active site of lysozyme, get affected by the presence of DMSO up to XDMSO ~ 0.2 [18]. The present CD measurements corroborate this simulation results. On further increase in the DMSO concentration the protein conformation changes as evidenced from the change in the spectral pattern coupled with a sharp drop in the intensity (Fig. 1b). CD study thus establishes conformational changes in the protein structure due to its specific interaction with DMSO. 3.2. FTIR studies As a control, we measure the FTIR spectra of the O-H stretching mode in water–DMSO binary mixtures (Fig. S1, Supporting information section). It is found that the intensity of the low frequency mode decreases with increasing DMSO concentration keeping the OH peak position unaltered. In addition, the full width at half maxima (FWHM) of the spectrum gets narrower, especially in the high DMSO concentration region. We deconvolute the O-H spectra into three Gaussian curves to quantify the relative contributions of different types of H-bonded water molecules: strongly H-bonded water or HW (appear in the lower frequency region), distorted water (DW) and isolated water (IW) (higher frequency region). HW is generally assigned to the water molecules having H-bonded coordination number close to four. Such a component closely relates the OH band observed in ice and this type of water is also denoted as the network water [35]. DW is defined as the water molecules which have the average degree of connections larger than that of the dimers or trimers but lower than those participating to the tetrahedral water networks. IW is defined as those water molecules which are poorly connected to their environments; mainly these are interfacial monomer waters or the water molecules which do not produce strong hydrogen bonds with neighboring water molecules [36]. Two representative deconvoluted spectra at XDMSO = 0 and XDMSO = 0.45 are shown in Fig. S2, (Supporting information section). The fit parameter for the deconvolution of HW, DW and IW are shown in Table S1. The O-H stretching spectrum of aqueous-HEWL solution is found to be comparable to that of pure water with only marginal changes in the HW and DW populations (Fig. S3, Supporting information section). However, as DMSO is added into the protein solution there occurs noticeable change in the absorption profile. A representative deconvolution at XDMSO = 0.45 is shown in Fig. S4 (Supporting information section). The fit parameter for the deconvolution of HW, DW and IW are also shown in Table S2. The width of the IW increases slightly with increasing DMSO concentration both in presence and in absence of the protein. It should be mentioned here that as IW molecules are poorly connected to their environments, they might not be fully isolated. However, it is also to be noted that the relative abundance of such molecules is very low and also does not change noticeably with
composition (Tables S1 and S2). So a reasonable discussion could be made on the basis of changes in HW and DW molecules only. Comparing Figs. S2 and S4, a striking dissimilarity is envisaged. We plot the relative abundance of HW and DW in absence and in presence of the protein (Fig. 2a,b). It can be noted here that the relative population of IW is very small and does not undergo noticeable change with DMSO. The HW and DW profile shows distinctly opposite trends in presence of the protein (Fig. 2). For the water–DMSO mixture the HW population decreases gradually with a concomitant increase in the DW population (the blue square symbols, Fig. 2). At low DMSO concentration (XDMSO b 0.1), the DMSO molecules tend to form micro-micellar aggregates involving 3–4 methyl moieties [10] which leads to a marginal change in the relative abundances. On further addition of DMSO, the micelle-like structures break up to produce extended structures involving extensive hydrogen bonding with water molecules. This in turn produces the observed sharp increase in the DW abundance at the expense of HW (Fig. 2). To explain the trends in the water–DMSO–HEWL ternary system one needs to consider the various interactions present: water–water, water–DMSO, water–protein as well as the protein–DMSO interactions. In water-HEWL binary solution the result can be described by considering the water–water and water–protein interactions only. As DMSO is introduced into the protein solution, it preferentially solvates the protein surface [16] expelling a fraction of the protein solvating water molecules into the bulk. This increases the relative abundance of water–water H-bond at the expense of water–DMSO H-bonds. The otherwise observed increase in the DW abundance in the binary mixture eventually reduces to a decrease in the DW abundance in presence of the protein (Fig. 2). Interaction of DMSO with HEWL changes the conformational flexibility as well as the solvent accessible surface area (SASA) of the protein [10]. One thus also has to additionally consider the protein–DMSO interaction which forms at the cost of water–DMSO interaction resulting in an overall decrease in the DW content. Beyond X DMSO ~ 0.3, the added DMSO molecules start interacting mostly with water and the DW abundance increases slightly. The FTIR study thus affirms the change in the hydration structure around HEWL as a result of the preferential solvation by DMSO. 3.3. TTDS study The THz absorption spectra for water–DMSO binary mixtures are shown in Fig. 3a. It can be noted here that absorption coefficient α(ν) in the THz frequency region is correlated with the collective dynamics of water and therefore is a useful parameter to study [20,27]. THz absorption decreases with increasing DMSO content; a closer insight reveals that the decrease is not linear. In order to obtain a more quantitative picture we plot α measured at 1 THz as a function of DMSO concentration (Fig. 3b). It is observed that α does not follow any linear trend with X DMSO , rather it passes through a minimum
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Fig. 2. Relative population of (a) hydrogen bonded water and (b) distorted water following the deconvolution (Gaussian) of MIR-FTIR spectra in water/DMSO/HEWL solutions at different DMSO concentrations. The broken lines are guide to the eyes.
indicating a non-ideal hydration behavior in the mixture. For an ideal mixture, the absorption coefficient (α id) obeys a two component model: α id ¼ α w φw þ α D φD
ð1Þ
where φ denotes the volume fraction. We plot αid in the same Fig. 3b (red dotted line) for comparison and observe marked deviation from ideality. To estimate the extent of deviation we plot Δα/αid (where Δα = α0-αid) as a function of XDMSO in the inset and observe a minimum at XDMSO ~ 0.25. It, therefore, is evident that a simple two component model is insufficient to account for the observed changes in α. Eq. (1) thus needs the introduction of a new term which takes care of the DMSO–water complex formation, α 0 ¼ α w φw þ α D φD þ α w−D φw−D
ð2Þ
where the last term appears due to the formation of water–DMSO complex structure. It is important to note that the dynamic hydration shell around solute molecules, which involves the ultrafast rearrangement in the associated hydrogen-bonded network, offers an absorption coefficient different from that of pure water [37]. The negative deviation from the ideality in α (Fig. 3b) can emanate from the term αw-D , or from a negative volume of mixing [38] or both. The negative excess molar volume of mixing (VEm) in DMSOwater mixture is indeed one of the responsible terms that contributes to the negative deviation in α. However, VEm has been reported to pass through a minimum at a relatively higher XDMSO ~ 0.4 [38], which, therefore indicates that hydration dynamics of the clustered structure in the mixture (as manifested by αw-D) does also contribute to the observed non-ideality.
With the background of anomalous water–DMSO interaction we now investigate the preferential solvation of the protein by DMSO. We measure the α(ν) profile of HEWL in DMSO–water solutions at different XDMSO. Some representative profiles are shown in Fig. 4a. In pure aqueous solution (uppermost panel) the absorption profile of the protein solution lies below that of pure water, which is explainable from the fact that water has high THz absorption than that of the proteins. However, a simple two component consideration is again insufficient to account for the observed change and for a more realistic rationale one should also consider the hydration sheathe of the protein, which offers different absorption coefficient than that of pure water [25,39]. The αobs(ν) thus cumulates contributions from bulk water, hydration water and protein [39]. It is now interesting to study how this hydration dynamics changes as DMSO induces the conformational changes. As discussed earlier, addition of DMSO invokes some additional pairwise interactions like water–DMSO, protein–DMSO and DMSO–DMSO. To compare the hydration structure of the protein we compare the absorption profile of the DMSO–water mixture at different compositions in absence and in presence of the protein. Some representative profiles are shows in the middle and lower panels of Fig. 4a. It is evident that the profiles change as XDMSO increases. At XDMSO = 0.05, the protein hydration curve lies well below the binary solution curve whereas at XDMSO = 0.25, it lies slightly above. In order to intrude into a more quantitative insight we plot the relative change in the absorption coefficient at 1 THz, denoted by Δα/α0 (where α0 is the absorption coefficient of the binary solution measured at 1 THz and Δα = αp-α0, where αp is the absorption coefficient measured at 1 THz in presence of the protein). The profile shows a minimum at XDMSO = 0.05 and then increases up to XDMSO = 0.15 beyond which it slowly decreases (Fig. 4b). The observation can be explained on the basis of the hydration around the protein as DMSO competes with water as a solvent. As DMSO is added into the protein solution, it replaces the hydrated
Fig. 3. (a) Frequency dependent Absorption coefficient α of water–DMSO binary mixture at different concentrations. The arrow indicates increasing DMSO concentration. (b) α measured at 1 THz for DMSO–water binary mixture as a function of DMSO concentration. The solid line is a guide to the eyes. The broken red line represents the ideal values of α. The inset shows the relative change in α.
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Fig. 4. (a) Absorption coefficient of water–DMSO mixtures in absence (blue, solid line) and in presence (red broken line) of HEWL. (b) Relative change in α of aqueous HEWL/DMSO solution as a function of DMSO concentration.
water (around protein) into the bulk and as a result the relative abundance of hydrated and bulk water changes. The preferential solvation of DMSO also in its turn disrupts the water-DMSO cluster structures. This effect is manifested in the changes in Δα. At XDMSO = 0.05, Δα decreases sharply and such a considerable change at a low DMSO content is intriguing. The major rationale behind this change perhaps emanates from the fact that the protein size increases by two folds at this DMSO concentration as evidenced by Ghosh et al. using FCS studies [19]. This in turn increases the effective volume fraction of the low absorbing protein molecules and consequently the overall Δα value decreases rapidly. As the concentration of DMSO increases further, the protein starts rebuilding its tertiary structure and the hydrodynamic radius approaches its native value [19]. During the process of refolding [18] the nonpolar solvent accessible surface area (SASA) of the protein increases leading to preferential accumulation of more DMSO molecules at the protein surface [16]. As a result of this preferential solvation the DMSO–water complex, otherwise present in the neat binary mixture, starts melting giving rise to more water–water hydrogen bonded network (compare DW and HW in Fig. 2a,b). The change in the relative volume fraction in presence and in absence of the protein induces an overall increase in the Δα at XDMSO = 0.1 compared to that in XDMSO = 0.05. Intriguingly, at XDMSO = 0.15, Δα has a positive value. The positive value of Δα at this composition can be rationalized from a relatively high positive contribution from the absorption of the protein hydration layer which overwhelms the otherwise negative contribution due to the preferential solvation of DMSO. It is important to note that at this composition the nonpolar SASA of the protein reaches its maximum [18], this in turn can change absorption due to the protein hydration layer and make the overall Δα positive. Further increase in the DMSO concentration decreases the nonpolar SASA [18], and also saturates the melting of the DMSO–water complex (see Fig. 2), which makes the Δα to decrease (Fig. 4b). 4. Conclusions Our study is aimed to understand whether the DMSO induced conformation changes in lysozyme conformation perturbs its hydration dynamics. CD study establishes a marked change in the protein tertiary structure in presence of DMSO. Preferential solvation of DMSO at the HEWL surface, as established from the MIR FTIR analysis, results in a relative decrease of DW population at the expense of HW content. The hydration dynamics at the protein surface is obtained from the relative change in the THz absorption coefficient (Δα/α0) which shows a negative minimum at XDMSO = 0.05 and a positive value at XDMSO = 0.15. The results are explained on the basis of the relative contributions of the absorption due to the various components present in the system.
The observed minimum is found to be due to the increased size of the protein while the positive value is attributed to the increased SASA and consequent increased hydration of the protein surface. Our study affirms that the collective hydration dynamics at the protein surface does follow the conformational changes in the protein caused by the preferential solvation of DMSO.
Acknowledgements RKM acknowledges DST, India for a research grant (SB/S1/PC-056/ 2013).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bpc.2016.06.002.
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