Archives of Biochemistry and Biophysics 573 (2015) 77–83
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Osmolyte mixtures have different effects than individual osmolytes on protein folding and functional activity Marina Warepam, Laishram Rajendrakumar Singh ⇑ Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India
a r t i c l e
i n f o
Article history: Received 15 September 2014 and in revised form 11 March 2015 Available online 26 March 2015 Keywords: Chemical denaturation Preferential hydration Synergistic effect Refolding efficiency Enzyme activity
a b s t r a c t Osmolytes are small organic molecules accumulated by organisms under stress conditions to protect macromolecular structure and function. In the present study, we have investigated the effect of several binary osmolyte mixtures on the protein folding/stability and function of RNase-A. For this, we have measured DGoD (Gibbs free energy change at 25 °C) and specific activity of RNase-A mediated hydrolysis of cytidine 20 –30 cyclic monophosphate in the presence and absence of individual and osmolyte mixtures. It was found that the osmolyte mixtures have different effect on protein stability and function than that of individual osmolytes. Refolding studies of RNase-A in the presence of osmolyte mixtures and individual osmolytes also revealed that osmolyte mixtures have a poor refolding efficiency relative to the individual osmolytes. Ó 2015 Published by Elsevier Inc.
Introduction Environmental stresses such as water stress, salt stress, cold and heat stress constantly challenge the stability and function of intracellular macromolecules [1–3]. One general mechanism of adaptation against these stresses is the accumulation of small molecular weight compounds known as osmolytes [1–3]. In general these osmolytes are grouped into three different chemical classes: polyols (e.g., sorbitol, sucrose, trehalose) commonly found in terrestrial plants, insects, polar fishes; free amino acids (e.g., proline, glycine, alanine) and their derivatives (e.g., taurine, ectonine, octopine) in numerous prokaryotes and methyl ammonium compounds (e.g., trimethylamine N-oxide, glycine betaine, sarcosine) in marine elasmobranches [1]. Based on functional activity they are grouped as (i) compatible osmolytes (that have no significant effect on enzyme activity e.g., most amino acids and polyols) [1,2,4] and (ii) non-compatible (that affects the enzyme activity e.g., all methylamines, lysine, arginine, histidine) [2,3,5]. The basic premise of accumulation of osmolytes is that they do not largely alter the enzyme’s functional activity even when present at a very high concentration (except some osmolytes like proline) [4] up to several millimolars [2,3]. The effect of osmolytes on protein stability and function has been largely investigated. It is well known that the ability of the osmolytes to fold or stabilize proteins is due to its unfavourable ⇑ Corresponding author. Fax: +91 11 27666248. E-mail address: [email protected] (L.R. Singh). http://dx.doi.org/10.1016/j.abb.2015.03.017 0003-9861/Ó 2015 Published by Elsevier Inc.
interactions with the peptide backbones exposed upon protein denaturation leading to preferential hydration of the protein molecules [6,7]. It has also been shown that (i) osmolytes can force folding of unstable, intrinsically disordered and many mutant proteins [8–10] and (ii) help to inhibit/reverse protein aggregation [11–13]. Therefore, osmolytes have been promising potential therapeutics for a large number of human diseases caused due to protein conformational disorders [14]. It is to be noted that almost all these developments were derived based on studies of the effect of individual osmolytes on proteins. In contrast, the cellular osmolytic pool generally consists of mixture of several osmolytes rather than individuals at a given stress condition [15–20]. Thus, results on the effect of individual osmolytes on protein folding bear less resemblances with that of the actual in vivo conditions. Therefore to have a more realistic protein folding insight in vitro by osmolytes, it is important to investigate protein folding and functional activity studies in the presence of different osmolyte mixtures (not with individual osmolytes). In the present study, we have investigated the effects of several binary osmolyte mixtures (found in the marine elasmobranches and mammalian kidney cells) on the activity and stability of Ribonuclease-A (RNase-A)1. We discovered that osmolyte mixtures have different effects than those of their individuals in protein stability and function. Osmolyte mixtures have poor protein folding ability (induces folded proteins that have low
1 Abbreviations used: Cm, the midpoint of denaturation; CD, circular dichroism; GdmCl, guanidinium chloride; e, molar absorption coefficient; Km, Michaelis constant; kcat, catalytic constant; RNase-A, Ribonuclease-A; TMAO, trimethylamine N-oxide.
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catalytic activity). The study indicates cross talks between/among osmolytes might help to regulate enzyme function in vivo. Materials and methods Commercially lyophilized preparation of RNase-A (type III-A) was purchased from Sigma Chemical Co. Betaine, sorbitol, TMAO, sarcosine, cytidine 20 –30 cyclic monophosphate (C > p) and 8-anilino-1-naphthalene sulfonic acid (ANS) were also obtained from Sigma. Guanidinium chloride (GdmCl) was purchased from MP Biomedicals. These chemicals, which were of analytical grade, were used without further purification. RNase-A solution was dialyzed extensively against 0.1 M KCl at pH 7.0 and 4 °C. Protein stock solution was filtered using 0.22 lm millipore filter paper. The protein gave a single band during polyacrylamide gel electrophoresis. Concentration of the protein solution was determined experimentally using molar absorption coefficient, e (M1 cm1) value of 9800 at 277.5 nm [21]. The concentration of GdmCl stock solution was determined by refractive index measurements [22]. All solutions for optical measurements were prepared in the degassed 0.05 M cacodylic acid buffer containing 0.1 M KCl. Since pH of the protein solution may change on the addition of cosolvents, pH of each solution was also measured after the denaturation and activity experiments. It was observed that the change in pH was not significant. Activity measurement of native RNase-A RNase-A activity was measured using cytidine 20 –30 cyclic monophosphate (C > p) as the substrate (0.25 mg ml1) following the procedure described by Crook et al. [23]. In order to see the effects of osmolytes on the activity of RNase-A, the enzyme (0.035 mg ml1) was titrated with the desired concentration of osmolytes and incubated overnight. The reaction for RNase-A mediated hydrolysis of C > p in the absence and presence of individuals and their binary osmolyte mixtures at a given concentration was followed by measuring absorbance at 292 nm for 1 h in Jasco V-660 UV/Vis spectrophotometer equipped with a Peltiertype temperature controller (ETCS-761). It should be noted that enzymatic reactions in the absence or presence of osmolytes were observed to be completed in 1 h. From each progress curve, the initial linear portion (usually first 60 s) was used as a measure of specific activity. GdmCl-induced denaturation measurements GdmCl-induced denaturation of native RNase-A in the presence and absence of individual osmolytes and their binary mixtures were followed by measuring changes in the [h]222 (mean residue ellipticity at 222 nm) as a function of GdmCl concentration at 25 °C and pH 7.0 using a J-810 (Jasco spectropolarimeter) equipped with a Peltier-type temperature controller (Jasco PTC-424S). Protein concentration used for circular dichroism was 0.5 mg/ml. It should be noted that each protein solution at each concentration of GdmCl was kept for adequate time (overnight) to allow equilibration. The CD signal was converted into mean residue ellipticity (deg cm2 dmol1) using the relation,
½hk ¼ hk Mo =10lc
ð1Þ
where hk is the observed ellipticity (millidegrees) at the wavelength k, Mo is the mean residue weight of the protein, c is the protein concentration (mg/cm3), l is the path length (centimetres). The optical transition data were converted into DGD, the Gibbs energy change for unfolding using the relation,
DGD ¼ RT lnfðy yN Þ=ðyD yÞg;
ð2Þ
where R is the universal gas constant, T is the temperature in Kelvin, y is the observed optical property and yN and yD are, respectively, the optical properties of the native and denatured protein molecules under the same experimental condition in which y has been determined. We then use linear extrapolation method for the analysis of DGoD as described previously [24,25], using the following equation
DGD ¼ DGoD md ½Gdmcl;
ð3Þ
DGoD
where is the value of DGD at 0 M denaturant, and md gives the linear dependence of DGD on the [GdmCl]. The reversibility of each of the GdmCl-induced denaturation curve was measured by diluting the protein samples at high GdmCl concentrations (5.5 M) down to the respective Cm values. It was observed that the optical property of the protein solution at Cm from the denaturation and renaturation experiments were similar. Activity measurement of refolded RNase-A For refolding experiments, heat-induced (85 °C) denatured RNase-A was cooled down in the presence of osmolytes to reach 25 °C using a dry bath (Indogenix). Refolding of the GdmCl-induced (5.5 M) denatured RNase-A was carried out by diluting the denatured protein to a ratio of l:100 using the cacodylic acid buffer (pH 7.0) that contains desired concentration of the osmolytes and kept overnight for equilibration. For the measurement of the specific activity of the refolded RNase-A, we used the same procedure described under the activity measurement of native RNase-A. All enzyme assays were carried out at least three times. Thermal-induced denaturation measurements Thermal-induced denaturation of native RNase-A and refolded RNase-A (from GdmCl, 5.5 M denatured state) in the presence and absence of binary osmolyte mixtures were followed by measuring changes in the [h]222 (mean residue ellipticity at 222 nm) as a function of temperature (25–85 °C) at pH 7.0 using a J-810 (Jasco spectropolarimeter) equipped with a Peltier-type temperature controller (Jasco PTC-424S) with a heating rate of 1 °C/min. This scan rate was found to provide adequate time for equilibration. Protein concentration used for circular dichroism was 0.5 mg/ml. After denaturation, the sample was cooled down to measure reversibility. The reversibility was checked by comparing the optical property of the protein before and after round of denaturation and was found to be identical (data not shown). Each heatinduced transition curve was analysed for Tm (the midpoint of heat denaturation) using a non linear least-squares analysis equation [26],
yðTÞ ¼
yN ðTÞ þ yD ðTÞ exp½DHm =Rð1=T 1=T m Þ 1 þ exp½DHm =Rð1=T 1=T m Þ
ð4Þ
where y(T) is the optical property at temperature T (Kelvin); yN(T) and yD(T) are the optical properties of the native and the denatured protein molecules, respectively; and R is the gas constant. In the analysis of the transition curve, it was assumed that a parabolic function describes the dependence of the optical properties of the native and denatured protein molecules i.e., yN(T) = aN + bNT + cNT2, and yD(T) = aD + bDT + cDT2, where aN, bN, cN, aD, bD, and cD are temperature-independent coefficients. Structural measurements Far- and near-UV CD spectra of native, refolded (obtained from GdmCl-induced denatured state) and denatured state of RNase-A
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were measured in the presence and absence of each 1 M individual and 1:1 M binary osmolyte mixtures at least three times in a J-810 (Jasco spectropolarimeter) equipped with a Peltier-type temperature controller (Jasco PTC-424S). Each spectrum of the protein was corrected for contribution of its blank in the entire wavelength range. The concentration of the protein used was 0.5 mg/ml. The path length of the cuvette used for far- and near-UV CD measurements were 1.0 and 10 mm respectively. It should be noted that the CD instrument was routinely calibrated with D-10-camphorsulfonic acid. Aggregation studies ANS-binding experiments ANS fluorescence measurements were made on a Perkin ElmerLS 55 (Fluorescence spectrometer). The fluorescence spectra of refolded RNase-A (from heat-induced denatured state) in the presence and absence of 1:1 M binary osmolyte mixtures were recorded from 380–600 nm using an excitation wavelength of 345 nm. The path length of the cuvette used for measurements was 5 mm. All necessary background corrections were made. Turbidity assay Turbidity of refolding process of RNase-A (85–25 °C) was measured by monitoring the change in optical density at 450 nm in the absence and presence of each 1:1 M binary osmolyte mixtures at pH 7.0 using a Jasco V-660 UV/Vis spectrophotometer equipped with a Peltier-type temperature controller (ETCS-761). The path length of the cuvette used for measurements was 10 mm. All necessary background corrections were made. Measurements were repeated for three times. Results The osmolytes chosen (betaine, sarcosine, TMAO and sorbitol) were based on the availability of these osmolytes in the osmoticum of various marine elasmobranches and mammalian kidney cells as mixtures [15,16,27]. We have made several mixtures that consist of two osmolytes (betaine + TMAO, betaine + sarcosine, TMAO + sarcosine, betaine + sorbitol) at equimolar ratio (i.e., 0.25:0.25, 0.5:0.5, 0.75:0.75 and 1.0:1.0). We have taken equimolar ratio as the cellular existence of any mixture ranges from 1:1 to 1:2 (except betaine + sarcosine) [28–30]. RNase-A was chosen as the protein is well characterised and several data on osmolyte-protein interactions exist in the literature. To investigate the possible effect of osmolyte mixtures on enzyme activity, we measured RNase-A mediated hydrolysis of cytidine 20 –30 cyclic monophosphate (C > p) in the absence and presence of different individuals and osmolyte mixtures at pH 7.0 and 25 °C. Representative time dependent curves of the RNase-A mediated hydrolysis of C > p in the presence of highest concentration of individual and osmolyte mixtures are shown in Fig. S1. Fig. 1 shows the effect of individual osmolytes and mixtures at different concentrations on the functional activity of RNase-A. It is seen in Figs. S1 and 1 that the effect of different osmolyte mixtures on the activity of RNase-A is different than that of the individuals. Fig. 2 shows the representative GdmCl-induced denaturation curves of RNase-A in the presence and absence of individual osmolytes and mixtures. It has been observed that yN (optical properties of native molecule) and yD (optical properties of denatured molecule) do not depend upon the type and concentration of the individual osmolytes and their mixtures. Each GdmCl-induced transition curve in the presence and absence of the osmolytes was analysed for estimating the thermodynamic unfolding parameters (DGoD and m-value) using Eq. (3). The evaluated
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thermodynamic parameters, DGoD, m-value and Cm, the midpoint of denaturation (= DGoD/md) are given in Table 1. It should, however, be noted that at 1.0:1.0 M concentration ratio for each of the osmolyte mixture, we could not obtain a complete GdmCl-induced transition curve due to experimental constraints (as enough GdmCl to denature the protein could not be added in the presence of 1.0:1.0 M of osmolyte mixtures), and hence yD was unknown. Since yD is independent of the [osmolyte], the molar concentration of osmolyte, we have analysed these incomplete transition curves by using property of yD for the control (in the absence of osmolyte). It is seen in Table 1 that individual osmolytes, and mixtures stabilize RNase-A in terms of Cm and DGoD. Fig. 3 shows the plot of DDGoD (DGoD of RNase-A in presence of osmolytes – DGoD of RNase-A in absence of osmolytes) versus [osmolyte]. It is also seen in this figure that osmolyte mixtures increase DDGoD of RNase-A more than the additive effects of the respective individuals on DGoD (at all osmolyte concentrations). We have further investigated the effect of the osmolyte mixtures on the refolding of the protein (from heat-induced denatured state) (Fig. 4). This figure clearly shows that refolding ability of the osmolyte mixtures are poor as compared to that of the individual osmolytes. ANS binding and turbidity measurements collectively indicate that there are no possibilities of protein aggregates formation in the presence of osmolyte mixtures (Fig. S3). In another experiment, we have performed osmolyte mixtures-induced refolding studies from GdmCl denatured state and compared it with that of the refolded proteins obtained from heat denatured state (see Fig. S4). It is seen in Fig. S4 that there is no significant difference in the specific activity of refolded native proteins obtained from heat- or GdmCl-induced denatured states. The results led us to believe that the reduction in the catalytic activity of the RNase-A is an intrinsic property of the osmolyte mixtures and does not depend on the type of the denaturing conditions. We then investigated if osmolyte mixtures induced folding of proteins to different extents as compared to that of the native protein (see Fig. S5). For this we have carried out measurements of secondary and tertiary structures of the refolded proteins (obtained from GdmCl denatured state) in the presence of osmolyte mixtures (Fig. S5). It is also seen in this figure that the structure of the refolded proteins are similar to that of the native protein (in absence of osmolytes). To further characterise the thermodynamic stability of folded protein molecules induced by osmolyte mixtures, we have performed thermal denaturation of the refolded protein molecules (folded in the presence of osmolyte mixtures from GdmCl-induced denatured state) (see Fig. S6). Each denaturation curve was analysed for the thermodynamic parameter Tm using Eq. (4). It was found that the stability of refolded RNase-A and native RNase-A in presence of osmolyte mixtures remains the same (see Fig. 5).
Discussion Our results on activity measurements of RNase-A suggest that osmolyte mixtures have different effect from that of the individuals. Indeed, individual osmolytes (except sorbitol) increases enzyme activity while the osmolyte mixtures do not largely affect the activity of RNase-A (see Fig. 1). In agreement to this result, the osmolytes chosen here (except sorbitol) have already been reported to increase enzyme activity (decrease Km or increase kcat) [31]. The methylamine osmolytes have been considered to be noncompatible with enzyme and cellular function [5,31,32]. Our results on the non-perturbance of enzyme activity in case of osmolyte mixtures suggest that in contrast to the effect of individual methylamine osmolytes, the mixtures might play a crucial role to make the non-compatible methylamine osmolytes compatible with enzyme function. Interestingly, methylamine osmolytes are
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Fig. 1. Effect of individual osmolytes and mixtures on RNase-A activity at pH 7.0 and 25 °C. Plot of specific activity (%) versus [osmolyte]. The percent specific activity of the enzyme in the absence of osmolytes was considered as 100%.
Fig. 2. Effect of individual osmolytes and mixtures on the GdmCl-induced unfolding profiles of RNase-A. Representative GdmCl-induced unfolding profiles of RNase-A in the presence of individual osmolytes and mixtures (at 1.0:1.0 M) at pH 7.0 and 25 °C.
known to exist as mixtures intracellularly [27,30,33]. It has been known that the osmolytes may affect the solvation properties of substrates or enzyme active sites or the physical properties of the solution thereby, affecting the enzyme substrate association and/or catalysis [4,31,34,35]. Therefore, the observed differential effects of the individual and osmolyte mixtures on enzyme activity might be due to having different effect on solvation properties of the substrates or enzyme active sites. It seems likely that osmolyte mixtures have little effect while individual osmolytes have large effect on the solvation properties of substrates or enzyme active sites. It is known that osmolytes stabilize proteins (or increase the number of N molecules) by shifting the denaturation equilibrium, N state M D state towards the left due to its unfavourable
osmophobic interaction with the peptide backbone (also called preferential hydration effect) resulting in an increase in the DGoD of the proteins [7,36]. Speculatively, the absence of a significant effect in the measured enzyme activity in the presence of osmolyte mixtures might be due to the fact that DGoD of the protein remains unperturbed in their presence. We are therefore, interested in investigating if protein stability (DGoD) is altered in the presence of the osmolyte mixtures. For this we performed GdmCl-induced denaturation of RNase-A in the presence and absence of different concentrations of each of the individual osmolyte and the mixture at pH 7.0 and 25 °C (Fig. 2). To analyse the denaturation curves for DGoD, three assumptions were made. First, it has been assumed that the GdmCl-induced denaturation of RNase-A in the absence and presence of different individual osmolytes and mixtures follows a
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M. Warepam, L.R. Singh / Archives of Biochemistry and Biophysics 573 (2015) 77–83 Table 1 Parameters characterising the GdmCl unfolding of RNase-A at pH 7.0 and 25 °C.a,b
DGoD (kcal/mol)
Cm (M)
m (kcal/mol M)
DGoD (kcal/mol)
Cm (M)
m (kcal/mol M)
DGoD (kcal/mol)
Cm (M)
m (kcal/mol M)
Control
10.3
3.0
3.4
10.3
3.0
3.4
10.3
3.0
3.4
[Osmolytes]
TMAO
0.25 M 0.50 M 0.75 M 1.00 M
10.7 10.8 11.2 11.8 (11.6)
3.4 3.6 3.9 4.1 (4.0)
3.5 3.8 3.6 3.7 (3.7)
3.2 3.4 4.0 4.3 (4.2)
3.5 3.4 3.3 3.4 (3.4)
3.4 3.5 3.9 4.2 (4.2)
3.5 3.9 3.7 3.6 (3.7)
3.3 3.4 3.6 4.2 (4.0)
3.5 3.7 3.8 3.5 (3.6)
Sarcosine 3.0 3.1 3.3 3.4 (3.4)
3.5 3.5 3.4 3.5 (3.4)
Betaine 0.25 M 0.50 M 0.75 M 1.00 M
10.8 11.2 11.7 12.1 (11.9)
10.8 11.2 11.7 12.1 (11.9)
3.0 3.1 3.4 3.6 (3.5)
3.6 3.6 3.4 3.4 (3.4)
3.0 3.1 3.4 3.6 (3.5)
3.6 3.6 3.4 3.4 (3.4)
10.8 11.2 11.7 12.1 (11.9)
3.5 3.6 3.5 3.5 (3.5)
3.0 3.1 3.4 3.6 (3.5)
3.6 3.6 3.4 3.4 (3.4)
10.7 10.8 11.2 11.8 (11.6)
11.2 11.7 12.2 12.5 (12.8)
3.0 3.1 3.3 3.4 (3.4)
3.5 3.5 3.4 3.5 (3.4)
11.5 11.7 13.3 14.6 (14.3)
3.2 3.2 3.5 3.6 (3.6)
3.5 3.6 3.5 3.5 (3.5)
12.0 13.6 14.4 15.2 (15.5)
3.0 3.1 3.3 3.4 (3.5)
3.6 3.5 3.5 3.5 (3.5)
Betaine + sarcosine
Sorbitol 10.8 11.0 11.4 11.9 (12.3)
11.9 13.9 14.2 15.2 (15.0) Betaine + TMAO
Sarcosine
Betaine 0.25 M 0.50 M 0.75 M 1.00 M
3.2 3.2 3.5 3.6 (3.6)
TMAO
Betaine 0.25 M 0.50 M 0.75 M 1.00 M
11.2 11.7 12.2 12.5 (12.8)
TMAO + sarcosine
Betaine + Sorbitol 11.7 12.7 13.7 14.8 (14.5)
a Values in the parentheses are from F305 measurements. Fluorescence measurements were made using a Horiba Jovin Yvon (Fluoromax-4, Spectrofluorometer) equipped with a Peltier-type temperature controller (LFI-3751), with an excitation wavelength of 268 nm. b Errors in DGoD, Cm and m from triplicate measurements are 3–6%, 1–1.5% and 1–2.5% respectively.
Fig. 3. Effect of osmolytes on protein stability. Plots showing DDGoD versus [osmolytes] of RNase-A in the presence of individuals and osmolyte mixtures. The expected additive line represents the sum total of DDGoD of the protein obtained in the presence of two respective individual osmolytes at various equimolar osmolyte concentrations.
two-state mechanism. This is indeed true in the absence of osmolytes [37]. In order to check whether a two-state assumption is also valid in the presence of individual osmolytes and mixtures, GdmClinduced denaturation curves of RNase-A were measured in the presence of highest concentrations of the individual osmolytes and mixtures using two different optical techniques at the same solution condition. One is by observing changes in the mean
residue ellipticity at 222 nm ([h]222) (see Fig. 2) and the other by measuring changes in the relative fluorescence (305 nm) (Fig. S2). We compared the values of DGoD and Cm obtained from the analysis of the curves using the two different probes. It has been observed that both measurements gave, within experimental errors, identical values of thermodynamic parameters (Table 1). Thus, the assumption that GdmCl-induced denaturation of
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Fig. 4. Effect of individual and binary osmolyte mixtures on the enzyme activity upon refolding from heat denatured (85 °C) RNase-A. Bar diagram of activity of refolded RNase-A in the presence of individual and osmolyte mixtures. The activity of the enzyme was estimated using 1.0 M of individual osmolytes and 1.0:1.0 M of their mixtures. The percent specific activity of the native enzyme in the absence of osmolytes was considered as 100%.
RNase-A in the presence of different individuals and osmolyte mixtures is a two-state process, is indeed valid. Another assumption made in the analysis of the GdmCl-induced transition curves of RNase-A in the presence of different individuals and osmolyte mixtures is that the DGD of RNase-A depends linearly on [GdmCl] under all experimental conditions. Indeed, it has already been shown that DGD of RNase-A in the presence of various individual osmolytes varies linearly with [GdmCl] throughout the denaturant concentration range [38–40]. Thus, it seems reasonable to assume that the linear dependence of DGD on [GdmCl] must be valid even in presence of the osmolyte mixtures. The third assumption is that the structural characteristics of the two end states of denaturation
Fig. 5. Effect of osmolyte mixtures on stability of refolded proteins. Bar diagram of DTm (Tm of RNase-A in presence of osmolytes – Tm of RNase-A in absence of osmolytes) of refolded RNase-A obtained from GdmCl-induced denatured state (white colour bar) and native RNase-A (grey colour bar) in the presence of 0.5:0.5 M osmolyte mixtures.
are not affected in the presence of individual and osmolyte mixtures. For this we have measured far- and near-UV CD spectra of both the native and denatured states in the presence and absence of individuals and osmolyte mixtures at pH 7.0 and 25 °C. It was observed that there is no significant alteration in the secondary and tertiary structures of the native protein and GdmCl-induced denatured state in the presence of the individual osmolytes and mixtures (see Fig. S5). This observation on the N-state of RNaseA is consistent with the X-ray diffraction results showing that the structure of the native state of proteins remains unperturbed by the addition of osmolytes [41] and with the size exclusion chromatography results which shows no alteration on the dimension of the native proteins in presence of osmolytes [36,42,43]. Our result on the denatured state of RNase-A is also in agreement with the result that shows no effect of osmolytes on the fully exposed amide protons [44]. Hence a comparison of a thermodynamic property of denaturation in the presence and absence of individuals and binary osmolyte mixtures is valid. The values of DGoD and Cm of RNase-A in the presence and absence of individual osmolytes shown in Table 1 are in close agreement with earlier reports [40,45]. It is seen in Table 1 and Fig. 3 that osmolyte mixtures show higher protein stabilizing (larger DGoD values) ability relative to that of the individual osmolytes. It appears that the mixtures have synergistic effect on the DGoD of RNase-A. Since preferential hydration induces protein folding and/or stabilization, the observed synergistic effect in the DGoD of RNase-A in the presence of osmolyte mixtures might be due to large increase in the preferential hydration effect as compared to the additive preferential effect of the two individual osmolytes. In contrast to the observed synergistic effect of osmolyte mixtures, it has been shown that urea and methylamine mixtures are thermodynamically additive [46]. The discrepancy might be because urea is a denaturing agent and perturbs water structure [47,48] while osmolytes are water structure enhancers [49]. Therefore, urea-methylamine can compensate each other in terms of the structure of water which is not the case in this study as we are using two stabilizing osmolytes. Taken together, the results of the effect of osmolyte mixtures on protein stability and enzyme activity indicate that osmolyte mixtures have different effect than individual osmolytes. The difference between the enzyme activities in presence of osmolyte mixtures and individual osmolytes may be due to the production of folded protein molecules with poor enzyme activity by the osmolyte mixtures. For this, we have intentionally refolded heat-induced denatured RNase-A (from 85 to 25 °C) in the presence of individual osmolytes and mixtures at pH 7.0 (see Fig. 4). Interestingly, we observed that there is 35–45% decrease in the enzyme activity in the presence of osmolyte mixtures while the activity in the presence of individual osmolytes remains unaltered. It has been previously reported that some osmolytes induces aggregate formation [50,51] that might be responsible for the reduction of enzyme activity. To confirm whether the decrease in the enzyme activity in the presence of osmolyte mixtures is due to formation of protein aggregates, we have performed ANS-binding experiment (see Fig. S3A). No increase in the ANS fluorescence in the presence of osmolyte mixtures indicates the absence of any protein aggregates during protein refolding process. This result is further confirmed by monitoring the refolding process of heat denatured RNase-A using light scattering intensity (see Fig. S3B). Detail characterizations of the refolded protein molecules (obtained from GdmCl denatured state) induced by osmolyte mixtures revealed that there is no significant difference in the structure and stability of the refolded proteins and with that of the native proteins in the presence of osmolyte mixtures indicating that the extent or magnitude of refolding is similar (see Figs. S5, 5 and S6). Taken together the results led us to believe that the
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decrease in enzyme activity in case of osmolyte mixtures is because of the production of stable protein molecules with poor catalytic enzyme activity. Speculatively, the production of stable native molecules with less catalytic activity by osmolyte mixtures might be due to the fact that osmolyte mixtures perhaps altered the folding pathway leading to the production of highly compacted native state that ultimately affects the flexibility of the active site of the enzyme molecule, and consequently making the enzyme difficult to pick substrates. Interestingly, many osmolytes at high concentrations have been reported to induce improper protein folding due to the production of stable near-native conformations that have reduced enzyme activity [31,52–55]. As osmolytes co-exist in the intracellular environment, the study alarms that protein folding in the cells may not be as simple as our current understanding on osmolyte-induced protein folding (with individual osmolytes) in vitro. Acknowledgments This work was supported by the grant from the Department of Science and Technology (Ref. No.: SR/SO/BB-0003/2011). LRS and MW acknowledge Council of Scientific and Industrial Research for the financial assistance provided in the form of research fellowship (File. no. 09/045(0992)/2010-EMR-1). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2015.03.017. References [1] P.H. Yancey, M.E. Clark, S.C. Hand, R.D. Bowlus, G.N. Somero, Science 217 (1982) 1214–1222. [2] P.H. Yancey, Sci. Prog. 87 (2004) 1–24. [3] P.H. Yancey, Biologist 50 (2003) 126–131. [4] A. Wang, D.W. Bolen, Biophys. J. 71 (1996) 2117–2122. [5] P.H. Yancey, G.N. Somero, Biochem J 183 (1979) 317–323. [6] S.N. Timasheff, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 9721–9726. [7] D.W. Bolen, I. Baskakov, J. Mol. Biol. 310 (2001) 955–963. [8] V.N. Uverski, J. Li, A.L. Fink, FEBS Lett. 509 (2001) 31–35. [9] P. Leandro, M.C. Lechner, I. Tavares de Almeida, D. Konecki, Mol. Genet. Metab. 73 (2001) 173–178. [10] L.R. Singh, X. Chen, V. Kozich, W.D. Kruger, Mol. Genet. Metab. 91 (2007) 335– 342. [11] D.S. Yang, C.M. Yip, T.H. Huang, A. Chakrabartty, P.E. Fraser, J. Biol. Chem. 274 (1999) 32970–32974.
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Osmolyte mixtures have different effects than individual osmolytes on protein folding and functional activity Marina Warepam, Laishram Rajendrakumar Singh ⇑ Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India
a r t i c l e
i n f o
Article history: Received 15 September 2014 and in revised form 11 March 2015 Available online 26 March 2015 Keywords: Chemical denaturation Preferential hydration Synergistic effect Refolding efficiency Enzyme activity
a b s t r a c t Osmolytes are small organic molecules accumulated by organisms under stress conditions to protect macromolecular structure and function. In the present study, we have investigated the effect of several binary osmolyte mixtures on the protein folding/stability and function of RNase-A. For this, we have measured DGoD (Gibbs free energy change at 25 °C) and specific activity of RNase-A mediated hydrolysis of cytidine 20 –30 cyclic monophosphate in the presence and absence of individual and osmolyte mixtures. It was found that the osmolyte mixtures have different effect on protein stability and function than that of individual osmolytes. Refolding studies of RNase-A in the presence of osmolyte mixtures and individual osmolytes also revealed that osmolyte mixtures have a poor refolding efficiency relative to the individual osmolytes. Ó 2015 Published by Elsevier Inc.
Introduction Environmental stresses such as water stress, salt stress, cold and heat stress constantly challenge the stability and function of intracellular macromolecules [1–3]. One general mechanism of adaptation against these stresses is the accumulation of small molecular weight compounds known as osmolytes [1–3]. In general these osmolytes are grouped into three different chemical classes: polyols (e.g., sorbitol, sucrose, trehalose) commonly found in terrestrial plants, insects, polar fishes; free amino acids (e.g., proline, glycine, alanine) and their derivatives (e.g., taurine, ectonine, octopine) in numerous prokaryotes and methyl ammonium compounds (e.g., trimethylamine N-oxide, glycine betaine, sarcosine) in marine elasmobranches [1]. Based on functional activity they are grouped as (i) compatible osmolytes (that have no significant effect on enzyme activity e.g., most amino acids and polyols) [1,2,4] and (ii) non-compatible (that affects the enzyme activity e.g., all methylamines, lysine, arginine, histidine) [2,3,5]. The basic premise of accumulation of osmolytes is that they do not largely alter the enzyme’s functional activity even when present at a very high concentration (except some osmolytes like proline) [4] up to several millimolars [2,3]. The effect of osmolytes on protein stability and function has been largely investigated. It is well known that the ability of the osmolytes to fold or stabilize proteins is due to its unfavourable ⇑ Corresponding author. Fax: +91 11 27666248. E-mail address: [email protected] (L.R. Singh). http://dx.doi.org/10.1016/j.abb.2015.03.017 0003-9861/Ó 2015 Published by Elsevier Inc.
interactions with the peptide backbones exposed upon protein denaturation leading to preferential hydration of the protein molecules [6,7]. It has also been shown that (i) osmolytes can force folding of unstable, intrinsically disordered and many mutant proteins [8–10] and (ii) help to inhibit/reverse protein aggregation [11–13]. Therefore, osmolytes have been promising potential therapeutics for a large number of human diseases caused due to protein conformational disorders [14]. It is to be noted that almost all these developments were derived based on studies of the effect of individual osmolytes on proteins. In contrast, the cellular osmolytic pool generally consists of mixture of several osmolytes rather than individuals at a given stress condition [15–20]. Thus, results on the effect of individual osmolytes on protein folding bear less resemblances with that of the actual in vivo conditions. Therefore to have a more realistic protein folding insight in vitro by osmolytes, it is important to investigate protein folding and functional activity studies in the presence of different osmolyte mixtures (not with individual osmolytes). In the present study, we have investigated the effects of several binary osmolyte mixtures (found in the marine elasmobranches and mammalian kidney cells) on the activity and stability of Ribonuclease-A (RNase-A)1. We discovered that osmolyte mixtures have different effects than those of their individuals in protein stability and function. Osmolyte mixtures have poor protein folding ability (induces folded proteins that have low
1 Abbreviations used: Cm, the midpoint of denaturation; CD, circular dichroism; GdmCl, guanidinium chloride; e, molar absorption coefficient; Km, Michaelis constant; kcat, catalytic constant; RNase-A, Ribonuclease-A; TMAO, trimethylamine N-oxide.
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catalytic activity). The study indicates cross talks between/among osmolytes might help to regulate enzyme function in vivo. Materials and methods Commercially lyophilized preparation of RNase-A (type III-A) was purchased from Sigma Chemical Co. Betaine, sorbitol, TMAO, sarcosine, cytidine 20 –30 cyclic monophosphate (C > p) and 8-anilino-1-naphthalene sulfonic acid (ANS) were also obtained from Sigma. Guanidinium chloride (GdmCl) was purchased from MP Biomedicals. These chemicals, which were of analytical grade, were used without further purification. RNase-A solution was dialyzed extensively against 0.1 M KCl at pH 7.0 and 4 °C. Protein stock solution was filtered using 0.22 lm millipore filter paper. The protein gave a single band during polyacrylamide gel electrophoresis. Concentration of the protein solution was determined experimentally using molar absorption coefficient, e (M1 cm1) value of 9800 at 277.5 nm [21]. The concentration of GdmCl stock solution was determined by refractive index measurements [22]. All solutions for optical measurements were prepared in the degassed 0.05 M cacodylic acid buffer containing 0.1 M KCl. Since pH of the protein solution may change on the addition of cosolvents, pH of each solution was also measured after the denaturation and activity experiments. It was observed that the change in pH was not significant. Activity measurement of native RNase-A RNase-A activity was measured using cytidine 20 –30 cyclic monophosphate (C > p) as the substrate (0.25 mg ml1) following the procedure described by Crook et al. [23]. In order to see the effects of osmolytes on the activity of RNase-A, the enzyme (0.035 mg ml1) was titrated with the desired concentration of osmolytes and incubated overnight. The reaction for RNase-A mediated hydrolysis of C > p in the absence and presence of individuals and their binary osmolyte mixtures at a given concentration was followed by measuring absorbance at 292 nm for 1 h in Jasco V-660 UV/Vis spectrophotometer equipped with a Peltiertype temperature controller (ETCS-761). It should be noted that enzymatic reactions in the absence or presence of osmolytes were observed to be completed in 1 h. From each progress curve, the initial linear portion (usually first 60 s) was used as a measure of specific activity. GdmCl-induced denaturation measurements GdmCl-induced denaturation of native RNase-A in the presence and absence of individual osmolytes and their binary mixtures were followed by measuring changes in the [h]222 (mean residue ellipticity at 222 nm) as a function of GdmCl concentration at 25 °C and pH 7.0 using a J-810 (Jasco spectropolarimeter) equipped with a Peltier-type temperature controller (Jasco PTC-424S). Protein concentration used for circular dichroism was 0.5 mg/ml. It should be noted that each protein solution at each concentration of GdmCl was kept for adequate time (overnight) to allow equilibration. The CD signal was converted into mean residue ellipticity (deg cm2 dmol1) using the relation,
½hk ¼ hk Mo =10lc
ð1Þ
where hk is the observed ellipticity (millidegrees) at the wavelength k, Mo is the mean residue weight of the protein, c is the protein concentration (mg/cm3), l is the path length (centimetres). The optical transition data were converted into DGD, the Gibbs energy change for unfolding using the relation,
DGD ¼ RT lnfðy yN Þ=ðyD yÞg;
ð2Þ
where R is the universal gas constant, T is the temperature in Kelvin, y is the observed optical property and yN and yD are, respectively, the optical properties of the native and denatured protein molecules under the same experimental condition in which y has been determined. We then use linear extrapolation method for the analysis of DGoD as described previously [24,25], using the following equation
DGD ¼ DGoD md ½Gdmcl;
ð3Þ
DGoD
where is the value of DGD at 0 M denaturant, and md gives the linear dependence of DGD on the [GdmCl]. The reversibility of each of the GdmCl-induced denaturation curve was measured by diluting the protein samples at high GdmCl concentrations (5.5 M) down to the respective Cm values. It was observed that the optical property of the protein solution at Cm from the denaturation and renaturation experiments were similar. Activity measurement of refolded RNase-A For refolding experiments, heat-induced (85 °C) denatured RNase-A was cooled down in the presence of osmolytes to reach 25 °C using a dry bath (Indogenix). Refolding of the GdmCl-induced (5.5 M) denatured RNase-A was carried out by diluting the denatured protein to a ratio of l:100 using the cacodylic acid buffer (pH 7.0) that contains desired concentration of the osmolytes and kept overnight for equilibration. For the measurement of the specific activity of the refolded RNase-A, we used the same procedure described under the activity measurement of native RNase-A. All enzyme assays were carried out at least three times. Thermal-induced denaturation measurements Thermal-induced denaturation of native RNase-A and refolded RNase-A (from GdmCl, 5.5 M denatured state) in the presence and absence of binary osmolyte mixtures were followed by measuring changes in the [h]222 (mean residue ellipticity at 222 nm) as a function of temperature (25–85 °C) at pH 7.0 using a J-810 (Jasco spectropolarimeter) equipped with a Peltier-type temperature controller (Jasco PTC-424S) with a heating rate of 1 °C/min. This scan rate was found to provide adequate time for equilibration. Protein concentration used for circular dichroism was 0.5 mg/ml. After denaturation, the sample was cooled down to measure reversibility. The reversibility was checked by comparing the optical property of the protein before and after round of denaturation and was found to be identical (data not shown). Each heatinduced transition curve was analysed for Tm (the midpoint of heat denaturation) using a non linear least-squares analysis equation [26],
yðTÞ ¼
yN ðTÞ þ yD ðTÞ exp½DHm =Rð1=T 1=T m Þ 1 þ exp½DHm =Rð1=T 1=T m Þ
ð4Þ
where y(T) is the optical property at temperature T (Kelvin); yN(T) and yD(T) are the optical properties of the native and the denatured protein molecules, respectively; and R is the gas constant. In the analysis of the transition curve, it was assumed that a parabolic function describes the dependence of the optical properties of the native and denatured protein molecules i.e., yN(T) = aN + bNT + cNT2, and yD(T) = aD + bDT + cDT2, where aN, bN, cN, aD, bD, and cD are temperature-independent coefficients. Structural measurements Far- and near-UV CD spectra of native, refolded (obtained from GdmCl-induced denatured state) and denatured state of RNase-A
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were measured in the presence and absence of each 1 M individual and 1:1 M binary osmolyte mixtures at least three times in a J-810 (Jasco spectropolarimeter) equipped with a Peltier-type temperature controller (Jasco PTC-424S). Each spectrum of the protein was corrected for contribution of its blank in the entire wavelength range. The concentration of the protein used was 0.5 mg/ml. The path length of the cuvette used for far- and near-UV CD measurements were 1.0 and 10 mm respectively. It should be noted that the CD instrument was routinely calibrated with D-10-camphorsulfonic acid. Aggregation studies ANS-binding experiments ANS fluorescence measurements were made on a Perkin ElmerLS 55 (Fluorescence spectrometer). The fluorescence spectra of refolded RNase-A (from heat-induced denatured state) in the presence and absence of 1:1 M binary osmolyte mixtures were recorded from 380–600 nm using an excitation wavelength of 345 nm. The path length of the cuvette used for measurements was 5 mm. All necessary background corrections were made. Turbidity assay Turbidity of refolding process of RNase-A (85–25 °C) was measured by monitoring the change in optical density at 450 nm in the absence and presence of each 1:1 M binary osmolyte mixtures at pH 7.0 using a Jasco V-660 UV/Vis spectrophotometer equipped with a Peltier-type temperature controller (ETCS-761). The path length of the cuvette used for measurements was 10 mm. All necessary background corrections were made. Measurements were repeated for three times. Results The osmolytes chosen (betaine, sarcosine, TMAO and sorbitol) were based on the availability of these osmolytes in the osmoticum of various marine elasmobranches and mammalian kidney cells as mixtures [15,16,27]. We have made several mixtures that consist of two osmolytes (betaine + TMAO, betaine + sarcosine, TMAO + sarcosine, betaine + sorbitol) at equimolar ratio (i.e., 0.25:0.25, 0.5:0.5, 0.75:0.75 and 1.0:1.0). We have taken equimolar ratio as the cellular existence of any mixture ranges from 1:1 to 1:2 (except betaine + sarcosine) [28–30]. RNase-A was chosen as the protein is well characterised and several data on osmolyte-protein interactions exist in the literature. To investigate the possible effect of osmolyte mixtures on enzyme activity, we measured RNase-A mediated hydrolysis of cytidine 20 –30 cyclic monophosphate (C > p) in the absence and presence of different individuals and osmolyte mixtures at pH 7.0 and 25 °C. Representative time dependent curves of the RNase-A mediated hydrolysis of C > p in the presence of highest concentration of individual and osmolyte mixtures are shown in Fig. S1. Fig. 1 shows the effect of individual osmolytes and mixtures at different concentrations on the functional activity of RNase-A. It is seen in Figs. S1 and 1 that the effect of different osmolyte mixtures on the activity of RNase-A is different than that of the individuals. Fig. 2 shows the representative GdmCl-induced denaturation curves of RNase-A in the presence and absence of individual osmolytes and mixtures. It has been observed that yN (optical properties of native molecule) and yD (optical properties of denatured molecule) do not depend upon the type and concentration of the individual osmolytes and their mixtures. Each GdmCl-induced transition curve in the presence and absence of the osmolytes was analysed for estimating the thermodynamic unfolding parameters (DGoD and m-value) using Eq. (3). The evaluated
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thermodynamic parameters, DGoD, m-value and Cm, the midpoint of denaturation (= DGoD/md) are given in Table 1. It should, however, be noted that at 1.0:1.0 M concentration ratio for each of the osmolyte mixture, we could not obtain a complete GdmCl-induced transition curve due to experimental constraints (as enough GdmCl to denature the protein could not be added in the presence of 1.0:1.0 M of osmolyte mixtures), and hence yD was unknown. Since yD is independent of the [osmolyte], the molar concentration of osmolyte, we have analysed these incomplete transition curves by using property of yD for the control (in the absence of osmolyte). It is seen in Table 1 that individual osmolytes, and mixtures stabilize RNase-A in terms of Cm and DGoD. Fig. 3 shows the plot of DDGoD (DGoD of RNase-A in presence of osmolytes – DGoD of RNase-A in absence of osmolytes) versus [osmolyte]. It is also seen in this figure that osmolyte mixtures increase DDGoD of RNase-A more than the additive effects of the respective individuals on DGoD (at all osmolyte concentrations). We have further investigated the effect of the osmolyte mixtures on the refolding of the protein (from heat-induced denatured state) (Fig. 4). This figure clearly shows that refolding ability of the osmolyte mixtures are poor as compared to that of the individual osmolytes. ANS binding and turbidity measurements collectively indicate that there are no possibilities of protein aggregates formation in the presence of osmolyte mixtures (Fig. S3). In another experiment, we have performed osmolyte mixtures-induced refolding studies from GdmCl denatured state and compared it with that of the refolded proteins obtained from heat denatured state (see Fig. S4). It is seen in Fig. S4 that there is no significant difference in the specific activity of refolded native proteins obtained from heat- or GdmCl-induced denatured states. The results led us to believe that the reduction in the catalytic activity of the RNase-A is an intrinsic property of the osmolyte mixtures and does not depend on the type of the denaturing conditions. We then investigated if osmolyte mixtures induced folding of proteins to different extents as compared to that of the native protein (see Fig. S5). For this we have carried out measurements of secondary and tertiary structures of the refolded proteins (obtained from GdmCl denatured state) in the presence of osmolyte mixtures (Fig. S5). It is also seen in this figure that the structure of the refolded proteins are similar to that of the native protein (in absence of osmolytes). To further characterise the thermodynamic stability of folded protein molecules induced by osmolyte mixtures, we have performed thermal denaturation of the refolded protein molecules (folded in the presence of osmolyte mixtures from GdmCl-induced denatured state) (see Fig. S6). Each denaturation curve was analysed for the thermodynamic parameter Tm using Eq. (4). It was found that the stability of refolded RNase-A and native RNase-A in presence of osmolyte mixtures remains the same (see Fig. 5).
Discussion Our results on activity measurements of RNase-A suggest that osmolyte mixtures have different effect from that of the individuals. Indeed, individual osmolytes (except sorbitol) increases enzyme activity while the osmolyte mixtures do not largely affect the activity of RNase-A (see Fig. 1). In agreement to this result, the osmolytes chosen here (except sorbitol) have already been reported to increase enzyme activity (decrease Km or increase kcat) [31]. The methylamine osmolytes have been considered to be noncompatible with enzyme and cellular function [5,31,32]. Our results on the non-perturbance of enzyme activity in case of osmolyte mixtures suggest that in contrast to the effect of individual methylamine osmolytes, the mixtures might play a crucial role to make the non-compatible methylamine osmolytes compatible with enzyme function. Interestingly, methylamine osmolytes are
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Fig. 1. Effect of individual osmolytes and mixtures on RNase-A activity at pH 7.0 and 25 °C. Plot of specific activity (%) versus [osmolyte]. The percent specific activity of the enzyme in the absence of osmolytes was considered as 100%.
Fig. 2. Effect of individual osmolytes and mixtures on the GdmCl-induced unfolding profiles of RNase-A. Representative GdmCl-induced unfolding profiles of RNase-A in the presence of individual osmolytes and mixtures (at 1.0:1.0 M) at pH 7.0 and 25 °C.
known to exist as mixtures intracellularly [27,30,33]. It has been known that the osmolytes may affect the solvation properties of substrates or enzyme active sites or the physical properties of the solution thereby, affecting the enzyme substrate association and/or catalysis [4,31,34,35]. Therefore, the observed differential effects of the individual and osmolyte mixtures on enzyme activity might be due to having different effect on solvation properties of the substrates or enzyme active sites. It seems likely that osmolyte mixtures have little effect while individual osmolytes have large effect on the solvation properties of substrates or enzyme active sites. It is known that osmolytes stabilize proteins (or increase the number of N molecules) by shifting the denaturation equilibrium, N state M D state towards the left due to its unfavourable
osmophobic interaction with the peptide backbone (also called preferential hydration effect) resulting in an increase in the DGoD of the proteins [7,36]. Speculatively, the absence of a significant effect in the measured enzyme activity in the presence of osmolyte mixtures might be due to the fact that DGoD of the protein remains unperturbed in their presence. We are therefore, interested in investigating if protein stability (DGoD) is altered in the presence of the osmolyte mixtures. For this we performed GdmCl-induced denaturation of RNase-A in the presence and absence of different concentrations of each of the individual osmolyte and the mixture at pH 7.0 and 25 °C (Fig. 2). To analyse the denaturation curves for DGoD, three assumptions were made. First, it has been assumed that the GdmCl-induced denaturation of RNase-A in the absence and presence of different individual osmolytes and mixtures follows a
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M. Warepam, L.R. Singh / Archives of Biochemistry and Biophysics 573 (2015) 77–83 Table 1 Parameters characterising the GdmCl unfolding of RNase-A at pH 7.0 and 25 °C.a,b
DGoD (kcal/mol)
Cm (M)
m (kcal/mol M)
DGoD (kcal/mol)
Cm (M)
m (kcal/mol M)
DGoD (kcal/mol)
Cm (M)
m (kcal/mol M)
Control
10.3
3.0
3.4
10.3
3.0
3.4
10.3
3.0
3.4
[Osmolytes]
TMAO
0.25 M 0.50 M 0.75 M 1.00 M
10.7 10.8 11.2 11.8 (11.6)
3.4 3.6 3.9 4.1 (4.0)
3.5 3.8 3.6 3.7 (3.7)
3.2 3.4 4.0 4.3 (4.2)
3.5 3.4 3.3 3.4 (3.4)
3.4 3.5 3.9 4.2 (4.2)
3.5 3.9 3.7 3.6 (3.7)
3.3 3.4 3.6 4.2 (4.0)
3.5 3.7 3.8 3.5 (3.6)
Sarcosine 3.0 3.1 3.3 3.4 (3.4)
3.5 3.5 3.4 3.5 (3.4)
Betaine 0.25 M 0.50 M 0.75 M 1.00 M
10.8 11.2 11.7 12.1 (11.9)
10.8 11.2 11.7 12.1 (11.9)
3.0 3.1 3.4 3.6 (3.5)
3.6 3.6 3.4 3.4 (3.4)
3.0 3.1 3.4 3.6 (3.5)
3.6 3.6 3.4 3.4 (3.4)
10.8 11.2 11.7 12.1 (11.9)
3.5 3.6 3.5 3.5 (3.5)
3.0 3.1 3.4 3.6 (3.5)
3.6 3.6 3.4 3.4 (3.4)
10.7 10.8 11.2 11.8 (11.6)
11.2 11.7 12.2 12.5 (12.8)
3.0 3.1 3.3 3.4 (3.4)
3.5 3.5 3.4 3.5 (3.4)
11.5 11.7 13.3 14.6 (14.3)
3.2 3.2 3.5 3.6 (3.6)
3.5 3.6 3.5 3.5 (3.5)
12.0 13.6 14.4 15.2 (15.5)
3.0 3.1 3.3 3.4 (3.5)
3.6 3.5 3.5 3.5 (3.5)
Betaine + sarcosine
Sorbitol 10.8 11.0 11.4 11.9 (12.3)
11.9 13.9 14.2 15.2 (15.0) Betaine + TMAO
Sarcosine
Betaine 0.25 M 0.50 M 0.75 M 1.00 M
3.2 3.2 3.5 3.6 (3.6)
TMAO
Betaine 0.25 M 0.50 M 0.75 M 1.00 M
11.2 11.7 12.2 12.5 (12.8)
TMAO + sarcosine
Betaine + Sorbitol 11.7 12.7 13.7 14.8 (14.5)
a Values in the parentheses are from F305 measurements. Fluorescence measurements were made using a Horiba Jovin Yvon (Fluoromax-4, Spectrofluorometer) equipped with a Peltier-type temperature controller (LFI-3751), with an excitation wavelength of 268 nm. b Errors in DGoD, Cm and m from triplicate measurements are 3–6%, 1–1.5% and 1–2.5% respectively.
Fig. 3. Effect of osmolytes on protein stability. Plots showing DDGoD versus [osmolytes] of RNase-A in the presence of individuals and osmolyte mixtures. The expected additive line represents the sum total of DDGoD of the protein obtained in the presence of two respective individual osmolytes at various equimolar osmolyte concentrations.
two-state mechanism. This is indeed true in the absence of osmolytes [37]. In order to check whether a two-state assumption is also valid in the presence of individual osmolytes and mixtures, GdmClinduced denaturation curves of RNase-A were measured in the presence of highest concentrations of the individual osmolytes and mixtures using two different optical techniques at the same solution condition. One is by observing changes in the mean
residue ellipticity at 222 nm ([h]222) (see Fig. 2) and the other by measuring changes in the relative fluorescence (305 nm) (Fig. S2). We compared the values of DGoD and Cm obtained from the analysis of the curves using the two different probes. It has been observed that both measurements gave, within experimental errors, identical values of thermodynamic parameters (Table 1). Thus, the assumption that GdmCl-induced denaturation of
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Fig. 4. Effect of individual and binary osmolyte mixtures on the enzyme activity upon refolding from heat denatured (85 °C) RNase-A. Bar diagram of activity of refolded RNase-A in the presence of individual and osmolyte mixtures. The activity of the enzyme was estimated using 1.0 M of individual osmolytes and 1.0:1.0 M of their mixtures. The percent specific activity of the native enzyme in the absence of osmolytes was considered as 100%.
RNase-A in the presence of different individuals and osmolyte mixtures is a two-state process, is indeed valid. Another assumption made in the analysis of the GdmCl-induced transition curves of RNase-A in the presence of different individuals and osmolyte mixtures is that the DGD of RNase-A depends linearly on [GdmCl] under all experimental conditions. Indeed, it has already been shown that DGD of RNase-A in the presence of various individual osmolytes varies linearly with [GdmCl] throughout the denaturant concentration range [38–40]. Thus, it seems reasonable to assume that the linear dependence of DGD on [GdmCl] must be valid even in presence of the osmolyte mixtures. The third assumption is that the structural characteristics of the two end states of denaturation
Fig. 5. Effect of osmolyte mixtures on stability of refolded proteins. Bar diagram of DTm (Tm of RNase-A in presence of osmolytes – Tm of RNase-A in absence of osmolytes) of refolded RNase-A obtained from GdmCl-induced denatured state (white colour bar) and native RNase-A (grey colour bar) in the presence of 0.5:0.5 M osmolyte mixtures.
are not affected in the presence of individual and osmolyte mixtures. For this we have measured far- and near-UV CD spectra of both the native and denatured states in the presence and absence of individuals and osmolyte mixtures at pH 7.0 and 25 °C. It was observed that there is no significant alteration in the secondary and tertiary structures of the native protein and GdmCl-induced denatured state in the presence of the individual osmolytes and mixtures (see Fig. S5). This observation on the N-state of RNaseA is consistent with the X-ray diffraction results showing that the structure of the native state of proteins remains unperturbed by the addition of osmolytes [41] and with the size exclusion chromatography results which shows no alteration on the dimension of the native proteins in presence of osmolytes [36,42,43]. Our result on the denatured state of RNase-A is also in agreement with the result that shows no effect of osmolytes on the fully exposed amide protons [44]. Hence a comparison of a thermodynamic property of denaturation in the presence and absence of individuals and binary osmolyte mixtures is valid. The values of DGoD and Cm of RNase-A in the presence and absence of individual osmolytes shown in Table 1 are in close agreement with earlier reports [40,45]. It is seen in Table 1 and Fig. 3 that osmolyte mixtures show higher protein stabilizing (larger DGoD values) ability relative to that of the individual osmolytes. It appears that the mixtures have synergistic effect on the DGoD of RNase-A. Since preferential hydration induces protein folding and/or stabilization, the observed synergistic effect in the DGoD of RNase-A in the presence of osmolyte mixtures might be due to large increase in the preferential hydration effect as compared to the additive preferential effect of the two individual osmolytes. In contrast to the observed synergistic effect of osmolyte mixtures, it has been shown that urea and methylamine mixtures are thermodynamically additive [46]. The discrepancy might be because urea is a denaturing agent and perturbs water structure [47,48] while osmolytes are water structure enhancers [49]. Therefore, urea-methylamine can compensate each other in terms of the structure of water which is not the case in this study as we are using two stabilizing osmolytes. Taken together, the results of the effect of osmolyte mixtures on protein stability and enzyme activity indicate that osmolyte mixtures have different effect than individual osmolytes. The difference between the enzyme activities in presence of osmolyte mixtures and individual osmolytes may be due to the production of folded protein molecules with poor enzyme activity by the osmolyte mixtures. For this, we have intentionally refolded heat-induced denatured RNase-A (from 85 to 25 °C) in the presence of individual osmolytes and mixtures at pH 7.0 (see Fig. 4). Interestingly, we observed that there is 35–45% decrease in the enzyme activity in the presence of osmolyte mixtures while the activity in the presence of individual osmolytes remains unaltered. It has been previously reported that some osmolytes induces aggregate formation [50,51] that might be responsible for the reduction of enzyme activity. To confirm whether the decrease in the enzyme activity in the presence of osmolyte mixtures is due to formation of protein aggregates, we have performed ANS-binding experiment (see Fig. S3A). No increase in the ANS fluorescence in the presence of osmolyte mixtures indicates the absence of any protein aggregates during protein refolding process. This result is further confirmed by monitoring the refolding process of heat denatured RNase-A using light scattering intensity (see Fig. S3B). Detail characterizations of the refolded protein molecules (obtained from GdmCl denatured state) induced by osmolyte mixtures revealed that there is no significant difference in the structure and stability of the refolded proteins and with that of the native proteins in the presence of osmolyte mixtures indicating that the extent or magnitude of refolding is similar (see Figs. S5, 5 and S6). Taken together the results led us to believe that the
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decrease in enzyme activity in case of osmolyte mixtures is because of the production of stable protein molecules with poor catalytic enzyme activity. Speculatively, the production of stable native molecules with less catalytic activity by osmolyte mixtures might be due to the fact that osmolyte mixtures perhaps altered the folding pathway leading to the production of highly compacted native state that ultimately affects the flexibility of the active site of the enzyme molecule, and consequently making the enzyme difficult to pick substrates. Interestingly, many osmolytes at high concentrations have been reported to induce improper protein folding due to the production of stable near-native conformations that have reduced enzyme activity [31,52–55]. As osmolytes co-exist in the intracellular environment, the study alarms that protein folding in the cells may not be as simple as our current understanding on osmolyte-induced protein folding (with individual osmolytes) in vitro. Acknowledgments This work was supported by the grant from the Department of Science and Technology (Ref. No.: SR/SO/BB-0003/2011). LRS and MW acknowledge Council of Scientific and Industrial Research for the financial assistance provided in the form of research fellowship (File. no. 09/045(0992)/2010-EMR-1). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2015.03.017. References [1] P.H. Yancey, M.E. Clark, S.C. Hand, R.D. Bowlus, G.N. Somero, Science 217 (1982) 1214–1222. [2] P.H. Yancey, Sci. Prog. 87 (2004) 1–24. [3] P.H. Yancey, Biologist 50 (2003) 126–131. [4] A. Wang, D.W. Bolen, Biophys. J. 71 (1996) 2117–2122. [5] P.H. Yancey, G.N. Somero, Biochem J 183 (1979) 317–323. [6] S.N. Timasheff, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 9721–9726. [7] D.W. Bolen, I. Baskakov, J. Mol. Biol. 310 (2001) 955–963. [8] V.N. Uverski, J. Li, A.L. Fink, FEBS Lett. 509 (2001) 31–35. [9] P. Leandro, M.C. Lechner, I. Tavares de Almeida, D. Konecki, Mol. Genet. Metab. 73 (2001) 173–178. [10] L.R. Singh, X. Chen, V. Kozich, W.D. Kruger, Mol. Genet. Metab. 91 (2007) 335– 342. [11] D.S. Yang, C.M. Yip, T.H. Huang, A. Chakrabartty, P.E. Fraser, J. Biol. Chem. 274 (1999) 32970–32974.
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