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Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins
Archives of Biochemistry and Biophysics 591 (2016) 7e17
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Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins Safikur Rahman 1, Syed Ausaf Ali, Asimul Islam, Md. Imtaiyaz Hassan, Faizan Ahmad* Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 August 2015 Received in revised form 4 November 2015 Accepted 21 November 2015 Available online 12 December 2015
Here we have generated two different denatured states using heat- and guanidinium chloride (GdmCl) induced denaturations of three disulfide bond free proteins (barstar, cytochrome-c and myoglobin). We have observed that these two denatured states of barstar and myoglobin are structurally and energetically different, for, heat-induced denatured state contains many un-melted residual structure that has a significant amount of secondary and tertiary interactions. We show that structural properties of the denatured state determine the magnitude of the protein stabilization in terms of Gibbs free energy change (DGD ) induced by an osmolyte, i.e., the greater the exposed surface area, the greater is the stabilization. Furthermore, we predicted the m-values (ability of osmolyte to fold or unfold proteins) using Tanford's transfer-free energy model for the transfer of proteins to osmolyte solutions. We observed that, for each protein, m-value is comparable with our experimental data in cases of TMAO (trimethylamine-N-oxide) and sarcosine. However, a significant discrepancy between predicted and experimental m-values were observed in the case of glycine-betaine. © 2015 Elsevier Inc. All rights reserved.
Keywords: Osmolytes Methylamines Protein stabilization Accessible surface area Gibbs free energy
1. Introduction Many proteins refold from their unfolded state to attain the native functional state. The correct folding of the protein polypeptide chain is determined not only by its amino acid sequence but also by the solvent environment. Cells are quite often exposed to widely fluctuating environmental conditions such as extremes of temperature, pH, cellular dehydration, desiccation, high extracellular salts, and even the presence of denaturing concentrations of urea inside the cell [1e3]. Protein folding in the cell is quite often challenged because of these environmental stresses. Failure to circumvent these hostile external environmental stresses may lead to various proteopathies, including protein misfolding, aggregation, amyloid formation and enhanced proteosomal degradation due to protein destabilization and eventually apoptosis [4,5]. Even the protein quality control system, including molecular chaperones, specialized intracellular proteases and accessory factors that
* Corresponding author. E-mail address: [email protected] (F. Ahmad). 1 Present address: Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India. http://dx.doi.org/10.1016/j.abb.2015.11.035 0003-9861/© 2015 Elsevier Inc. All rights reserved.
regulate the activity of chaperones and proteases, may also get damaged due to the extreme stresses. Interestingly, organisms or cells have a developed mechanism of accumulation of low molecular mass organic molecules called osmolytes which protect them against adverse environmental stresses. Osmolytes can be grouped into three chemical classes as polyols and sugars (e.g., trehalose, glycerol, inositols, etc.), amino acids (e.g., glycine, proline, etc.) and their derivatives (e.g., balanine, taurine, etc.) and combinations of methylamines (e.g., sarcosine, trimethylamine-N-oxide (TMAO), etc.) and urea [3,6]. Often, they are classified as compatible or counteracting based on their effects on both the stability and function of proteins [7]. Compatible osmolytes increase stability of proteins against thermal denaturation with little or no effect on their function near room temperature [8,9]. Representatives of this class include certain amino acids and polyols [8,9]. Counteracting osmolytes, on the other hand, are believed to have a special ability to protect intracellular proteins against inactivation and/or destabilization by urea [10e13]. That is, they cause changes that are opposite to the effect of urea on protein stability and function of the protein [1,9,14,15]. Organs like mammalian kidney and many animals like cartilaginous fishes and coelacanth that are rich in urea, employ these
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counteracting osmolytes including glycine-betaine, glycerophosphorylcholine (GPC) and TMAO [6,8,10]. There are two defining characteristics of compatible osmolytes, namely, they stabilize proteins against denaturation by heat or chemicals [16e21], and their presence in the cell does not largely alter protein functional activity [3,9,18,22e24]. The mechanism of osmolytes-induced protein stability has been studied extensively [3,9,25e28]. It has been widely accepted that osmolytes stabilize or induce protein folding due to preferential hydration effect [26,27]. Bolen et al. [29,30] from their elegant series of experiments that measured the transfer-free energy of the amino acid side chains and backbone, showed that the preferential hydration effect on proteins is chiefly because of the unfavorable interactions of osmolytes with the peptide backbone in the denatured state while the total contribution of amino acid side chains is little. Their studies led to the conclusion that osmolytes-induced thermodynamic stability of proteins is due to shift in denaturation equilibrium, N (native state) 4 D (denatured state), from right to the left. Although, osmolytes-facilitated protein stability has been deeply studied, most of these studies have been carried out on proteins having disulfide bond cross links or intrinsically unstructured proteins. Furthermore, to the best of our knowledge, no earlier studies compared osmolyte-induced thermodynamic stability of a protein obtained from heat- and GdmCl-induced denaturation, as it is widely known that these two modes of protein denaturation may give structurally two different denatured states [31e33]. To mimic the intercellular state of disulfide bonds containing proteins, we have used three different disulfide free proteins. We report that, for such a protein, the osmolyte-induced stabilizing effect in terms of Gibbs free energy change (DGD ), obtained from the GdmCl-induced denaturation is larger than that obtained from the heat-induced denaturation. Furthermore, we have theoretically predicted the m-value (ability of osmolyte to refold or unfold protein) and found a good correlation between theoretical and experimental m-values of all GdmCl-denatured proteins.
2. Materials and methods 2.1. Materials Commercial lyophilized preparation of horse heart myoglobin (Mb) and bovine cytochrome-c (b-cyt-c) were purchased from Sigma Chemical Co (St. Louis, MO). Barstar (from Bacillus amyloliquefaciens) was expressed in Escherichia coli and purified by the method published by Khurana and Udgaonkar [31]. GdmCl was purchased from MP Biomedical Inc. TMAO, Sarcosine and Glycinebetaine (GB) were obtained from Sigma Chemical Co (St. Louis, MO). These and other chemicals, which were of analytical grade, were used without further purification.
2.2. Preparation of protein solutions Mb and b-cyt-c were oxidized by dissolving them in 0.1 and 0.01% potassium ferricyanide solutions, respectively. These solutions were then dialyzed against several changes of 0.1 M KCl solution (pH 7.0) at 4 C. Barstar was also dialyzed in cold against 0.1 M KCl at pH 7.0. The concentrations of stock solutions of proteins were determined using molar absorption coefficient, ε (M1cm1) values of 171,000 at 409 nm for Mb [34], 106,000 at 409 nm for bcyt-c [35] and 23,000 at 280 nm for barstar [31]. All solutions for optical measurements were prepared in the desired degassed buffer containing 0.1 M KCl.
2.3. Spectroscopic determination of free energy change associated with GdmCl- and heat-induced denaturations GdmCl-induced denaturations of b-cyt-c, Mb and barstar in the absence and presence of different concentrations of each methylamine were monitored by following changes in [q]222 (molar ellipticity at 222 nm) at pH 7.0 and 25 C. The unfolding transitions of these proteins were reversible in the entire [g] (molar GdmCl concentration) range in the presence of each co-solute. Using a non-linear least-squares method, the entire data (y(g), [g]) of each denaturant-induced transition curve were analyzed for DGD , mg and Cm using the relation [36],
yðgÞ ¼
0 yN ðgÞ þ yD ðgÞ e½ðDGD þmg ½gÞ=RT 0 1 þ e½ðDGD þmg ½gÞ=RT
(1)
where y(g) is the observed optical property at [g], yN and yD are optical properties of the native and denatured protein molecules under the same experimental conditions in which y(g) was measured, DGD is the value of Gibbs free energy change (DGD) in the absence of denaturant, mg is the slope (vDGD/v[g])T,P, R is the universal gas constant, and T is the temperature in Kelvin. It should, however, be noted that the analysis of each GdmCl-induced transition curve was done assuming that unfolding is a two-state process and [g]-dependencies of yN(g) and yD(g) are linear (i.e., yN(g) ¼ aN þ bN [g] and yD(g) ¼ aD þ bD [g], where a and b are [g]independent parameters, and subscripts N and D represent these parameters for the native and denatured protein molecules, respectively). Heat-induce-denaturation studies were carried out in Jasco spectropolarimeter, Model J-1500-150 (JASCO Corporation, Japan), equipped with Peltier-type temperature controller, at a heating rate of 1 C/min. This scan rate was found to provide adequate time for equilibration. The change in secondary structure of the protein with increasing temperature was followed by measuring the far-UV CD at 222 nm. About 650 data points (data point at 0.1 C interval) of each transition curve were collected. After denaturation, the sample was immediately cooled down to measure reversibility of the process. Each heat-induced transition curve was analyzed for Tm and DHm using a non-linear least-squares analysis according to the relation:
yðTÞ ¼
yN ðTÞ þ yD ðTÞ exp ½ DHm =Rð1=T 1=Tm Þ 1 þ exp½ DHm =Rð1=T 1=Tm Þ
(2)
where y(T) is the optical property at temperature T (Kelvin), yN(T) and yD(T) are the optical properties of the native and denatured protein molecules at temperature T (Kelvin), and R is the gas constant. In the analysis of each heat-induced 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) ¼ aN þ bNT þ cNT2 and yD(T) ¼ aD þ bDT þ cDT2, where aN, bN, cN, aD, bD, and cD are temperature-independent coefficients) [37,38]. The value of the temperature-independent constant-pressure heat capacity change (DCp) was determined from the slope of the linear plots of DHm versus Tm, using the relation:
DCp ¼
dDHm dTm
(3) P
Using values of Tm, DHm and DCp, the value of DGD at any temperature T, DGD(T) was estimated using the GibbseHelmholtz equation:
S. Rahman et al. / Archives of Biochemistry and Biophysics 591 (2016) 7e17
DGD ðTÞ ¼ DHm
Tm T Tm
T DCp ðTm TÞ þ T ln Tm
(4)
2.4. Estimation of m-values using Tanford's transfer-free energy model [39] The program DSSP [40] was used to estimate accessible surface area (ASA) of proteins using PDB coordinates of 1YMB (Mb from horse heart), 1BTA (barstar from Bacillus amyloliquefaciens) and 2B4Z (b-cyt-c from bovine heart). Transfer-free energy values from water to 1.0 M osmolyte solution for side chains and peptide
9
backbone were used as given by Auton and Bolen [41] who showed that the total transfer-free energy of a protein is related to mos (ability of osmolyte to unfold or re-fold the protein) through the relation:
mos ¼
X
ni Dai Dgtr;i
(5)
where ni is the number of the ith type of residue (eNHeCHReCOe) in the protein, Dgtr,i is the transfer-free energy change of the ith type of residue when it is transferred from water to the 1.0 M osmolyte solution, and Dai is the fractional increase in the exposure of the ith type of residue on denaturation. Dai was calculated using the relation:
Fig. 1. GdmCl-induced denaturation curves of proteins in the presence and absence of osmolytes at pH 7.0 and 25 C. GdmCl-induced denaturation profiles of Mb (A), barstar (B) and b-cyt-c (C) in the absence and presence of methylamines. Control (ο) represents denaturation curve in the absence of osmolytes. Symbols (D), (7) and ( ) represent 0.5 M sarcosine, 0.5 M TMAO and 0.5 M GB, respectively, while (:), (;) and (-) represent1.0 M TMAO, 1.0 M sarcosine and 1.0 M GB, respectively. Far-UV CD spectra of the native (solid line), GdmCl denatured in 3.0 M GdmCl (line-dot) and renatured Mb (D), barstar (E) and b-cyt-c (F) in 1.0 M GdmCl (long dash).
▫
10
Dai ¼
S. Rahman et al. / Archives of Biochemistry and Biophysics 591 (2016) 7e17
P XASA;i FASA;i XASA;i
(6)
XASA,i and FASA,i are accessible surface areas of the ith type of residue in the denatured and native states, respectively. Using DSSP server and PDB coordinates, we calculated FASA,i value of each type of residue in the native state. For estimation of XASA,i we have used denatured state model of Bernado et al. [42]. 3. Results 3.1. Protein stability from heat and GdmCl-induced denaturation studies To investigate the interaction of osmolyte with heat-induced and GdmCl-induced denatured states, we have intentionally chosen three proteins (Mb, barstar and b-cyt-c) which do not contain disulfide bond. To estimate the effect of osmolytes on DGD (at 25 C) of these proteins, we measured heat-induced and GdmClinduced denaturation curves in the presence of various concentrations (0, 0.25, 0.50, 0.75, and 1.0 M) of each of the methylamines (sarcosine, TMAO and GB) by following changes in [q]222 (a probe for change in secondary structure). Denaturation (heat- or GdmClinduced) of each of these proteins was reversible in the entire range of [osmolyte], the molar concentration of the methylamine. Fig. 1 shows GdmCl-induced denaturation curves of Mb, barstar and bcyt-c in the presence of 0, 0.5 and 1.0 M osmolytes at pH 7.0. Results at other concentrations of each osmolyte are shown elsewhere (see Figure 1 in ref. [#]). It was observed that for each protein the dependence of yN and yD on [g], the molar concentration of GdmCl, is independent of the osmolyte type and its concentration (see Fig. 1 and (Fig. 1 in ref. [#])). Furthermore, it was observed that, for each protein, yN does not depend on [g]. However, yD of Mb and bcyt-c showed slight dependence on [g]. Each transition curve, which was measured at least three times, was analyzed for thermodynamic parameters using Eq. (1). Values of DGD , mg and Cm thus obtained are given in Table 1. Fig. 2 shows heat-induced denaturation curves of Mb, barstar and b-cyt-c in the presence of 0, 0.5 and 1.0 M sarcosine, TMAO and GB at pH 7.0. Fig. 2 in ref. [#] shows denaturation curves at other concentrations of each osmolytes at pH 7.0. Figs. 3e5 in the ref. [#] show denaturation curves of these proteins in the presence of 0,
0.25, 0.50 and 1.0 M osmolyte at pH values other than 7.0. Each transition curve of a protein at a given [osmolyte] and pH was analyzed for thermodynamic parameters (Tm and DHm) using a non-linear least-squares method that involves fitting the entire ([q]222, T) data of the transition curve to Eq. (2) with all eight free parameters (aN, bN, cN, aD, bD, cN, Tm and DHm). Values of Tm and DHm of all three proteins in the presence and absence of different osmolytes at pH 7.0 are given in Table 2. The values of these parameters of proteins in the absence and presence of different concentrations of each osmolyte at pH values other than pH 7.0 are given in Tables 1e3 in ref. ([#]). It may be noted that for heatinduced denaturations, we were not able to obtain complete transition curves in cases of Mb and b-cyt-c. So in order to bring down the thermal transition curves in the measurable range, we carried out denaturation studies in the presence of 0.6 and 1.25 M GdmCl for Mb and b-cyt-c, respectively. To correct for the contribution of GdmCl to the observed Tm and DHm, we carried out measurements of denaturation curves of these proteins in the presence of three more GdmCl concentrations (0.4, 0.8 and 1.0 M for Mb; and 0.6, 1.0 and 1.5 M for b-cyt-c) at different pH values. The results of such measurements of pH 7.0 are shown in Fig. 3. Corrections were done based on the method published earlier [21]. These corrected values of Tm and DHm are entered in Table 2 and Tables 1 and 2 in Ref. [Ref. #]. To estimate DCp of a protein at a given [osmolyte], DHm values were determined at number of Tm values by varying the pH, and the slope of the straight line of the DHm versus Tm plot is used to estimate DCp using Eq. (3) [43]. Values of DHm and Tm at different osmolyte concentrations and pH values are given in Table 2 and Tables 1e3 in ref. [#]. It should be noted that for a protein at a given [osmolyte], all 15 (DHm, Tm) values obtained from triplicate measurements at all pH values were used to construct the DHm versus Tm plot to determine DCp. Table 3 shows values of DCp of all proteins in the presence of different concentrations of sarcosine, TMAO and GB. It is seen in this table that DCp of proteins does not depend on [osmolyte]. At a constant pH and [osmolyte], DGD , the value of DGD in the absence of GdmCl at 25 C, was estimated using Eq. (4) with known values of DHm, Tm and DCp. However, this estimation requires a large extrapolation. Hence, a large error may be associated with DGD determination due to errors in the estimations of DHm, Tm, and DCp. We have used Becktel and Schellman's procedure [43] to
Table 1 Thermodynamic parameters associated with the GdmCl-induced denaturation of Mb, barstar and b-cyt-c in the absence and presence of different concentrations of osmolytes at pH 7.0 and 25 C. [Osmolytes] M
Sarcosine 0.00 0.25 0.50 0.75 1.00 TMAO 0.25 0.50 0.75 1.00 GB 0.25 0.50 0.75 1.00
Mb
Barstar
b-cyt-c
DGD
mg
Cm
DGD
mg
Cm
DGD
mg
Cm
kcal mol1
kcal mol1 M1
M
kcal mol1
Kcal mol1 M1
M
kcal mol1
kcal mol1 M1
M
7.08 8.01 8.98 10.01 10.85
± ± ± ± ±
0.26 0.25 0.24 0.22 0.21
4.43 4.42 4.51 4.47 4.49
± ± ± ± ±
0.10 0.07 0.09 0.10 0.12
1.60 1.81 1.99 2.23 2.42
± ± ± ± ±
0.06 0.09 0.06 0.09 0.09
4.98 5.48 5.89 6.61 7.35
± ± ± ± ±
0.21 0.16 0.21 0.18 0.20
2.63 2.62 2.58 2.53 2.59
± ± ± ± ±
0.09 0.04 0.05 0.10 0.08
1.89 2.09 2.28 2.61 2.83
± ± ± ± ±
0.11 0.06 0.09 0.08 0.08
10.60 11.33 12.02 12.78 13.20
± ± ± ± ±
0.21 0.25 0.27 0.28 0.24
3.99 4.03 4.04 4.06 3.98
± ± ± ± ±
0.06 0.0.8 0.10 0.07 0.04
2.66 2.81 2.97 3.15 3.31
± ± ± ± ±
0.15 0.12 0.14 0.13 0.12
8.09 9.04 10.29 11.34
± ± ± ±
0.23 0.17 0.22 0.29
4.39 4.36 4.50 4.51
± ± ± ±
0.07 0.08 0.04 0.07
1.85 2.08 2.29 2.52
± ± ± ±
0.10 0.11 0.06 0.08
5.53 6.14 6.74 7.51
± ± ± ±
0.17 0.24 0.13 0.21
2.68 2.76 2.56 2.65
± ± ± ±
0.08 0.09 0.07 0.08
2.06 2.22 2.63 2.83
± ± ± ±
0.09 0.07 0.07 0.09
11.27 12.31 12.98 13.39
± ± ± ±
0.28 0.25 0.24 0.29
4.01 4.17 4.06 3.97
± ± ± ±
0.10 0.08 0.09 0.10
2.81 2.95 3.19 3.37
± ± ± ±
0.14 0.12 0.15 0.10
7.95 8.42 9.39 10.14
± ± ± ±
0.19 0.21 0.27 0.25
4.42 4.52 4.55 4.44
± ± ± ±
0.09 0.07 0.06 0.08
1.80 1.86 2.06 2.28
± ± ± ±
0.09 0.05 0.07 0.08
5.54 5.94 6.72 7.24
± ± ± ±
0.17 0.24 0.18 0.21
2.54 2.55 2.69 2.68
± ± ± ±
0.04 0.08 0.07 0.05
2.18 2.33 2.50 2.70
± ± ± ±
0.10 0.07 0.14 0.06
11.23 12.03 12.53 12.92
± ± ± ±
0.19 0.29 0.35 0.15
4.04 4.02 3.89 3.85
± ± ± ±
0.09 0.10 0.11 0.05
2.78 2.99 3.22 3.35
± ± ± ±
0.12 0.09 0.10 0.12
S. Rahman et al. / Archives of Biochemistry and Biophysics 591 (2016) 7e17
11
Fig. 2. Heat-induced denaturation curves of proteins at pH 7.0. Denaturation profiles of Mb (A), barstar (B) and b-cyt-c (C) in the absence and presence of methylamines. Denaturation curves in cases of Mb and b-cyt-c were obtained in the presence of 0.6 and 1.25 M GdmCl, respectively. Symbols have same meaning as in Fig. 1. To maintain clarity all data points are not shown. Far-UV CD spectra of the native (solid line), heat-denatured at 85 C (line-dot) and renatured Mb (D), barstar (E) and b-cyt-c (F) at 20 C (long dash).
determine the maximum and minimum errors associated with the DGD determination in a given solvent condition. As there were three independent measurements of DHm and Tm of a protein at the given pH and [osmolyte], we obtained six values of DGD (three maximum and three minimum values). All of these six values were used to determine the average DGD and the mean error. The average values of DGD of all proteins at pH 7.0 are given in Table 2. Tables 1e3 in ref. [#] show the average values at other pH values. 3.2. Far-UV CD properties of denatured states Is there a structural difference between the GdmCl- and heatinduced denatured states of a protein? To answer this question, the far-UV CD (measure of change in secondary structure) measurements were done for all proteins in the presence of 4.0 M
GdmCl at 25 C and at 85 C (see Fig. 4). It may, however, be noted that the heat-denatured states of b-cyt-c and Mb obtained here already contained 1.25 M and 0.6 M GdmCl respectively, as heatinduced denaturation in the absence of the chemical denaturant is not complete at 85 C. Therefore, it is important to know whether the chemical denaturant has any effect on the heat-denatured state of these two proteins. For this, the far-UV CD spectra of the denatured b-cyt-c and Mb in the presence of different concentrations of GdmCl (0.4, 0.6, 0.8 and 1.0 for Mb and 0.6, 1.0, 1.25, 1.5 for b-cyt-c) were measured at 85 C (see Fig. 6 in [ref. #]). Values of [q] at different wavelengths were read from each spectrum measured in a given concentration of the chemical denaturant. Values of [q] at each wavelength were plotted as a function of [GdmCl] (e.g., see Fig. 4, inset). It was observed that the plot of [q]l versus [GdmCl] is linear, and a linear extrapolation of this plot to 0 M of the chemical
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S. Rahman et al. / Archives of Biochemistry and Biophysics 591 (2016) 7e17
Table 2 Thermodynamic parameters associated with the thermal denturation of Mb, barstar and b-cyt-c in the absence and presence of different concentrations of osmolytes at pH 7.0. [Osmolytes] M
Mb
Barstar
DGD kcal mol Sarcosine 0.00 0.25 0.50 0.75 1.00 TMAO 0.25 0.50 0.75 1.00 GB 0.25 0.50 0.75 1.00
1
C
b-cyt-c
DGD
DHm
Tm
kcal mol
1
kcal mol
1
DGD
DHm
Tm C
kcal mol
1
kcal mol
Tm
D Hm
1
kcal mol1
C
7.10 7.41 7.75 7.99 8.35
± ± ± ± ±
0.19 0.16 0.34 0.35 0.21
85.5 86.3 88.2 90.4 91.9
± ± ± ± ±
0.3 0.3 0.4 0.2 0.3
121 122 124 126 127
± ± ± ± ±
2 2 3 3 3
4.55 4.82 5.27 5.59 5.95
± ± ± ± ±
0.23 0.19 0.17 0.22 0.21
72.0 72.4 73.3 74.7 75.8
± ± ± ± ±
0.3 0.1 0.2 0.2 0.4
65 66 70 72 74
± ± ± ± ±
2 3 3 4 3
10.60 11.24 11.72 12.42 12.98
± ± ± ± ±
0.23 0.28 0.16 0.27 0.21
86.6 88.5 90.0 92.3 93.8
± ± ± ± ±
0.4 0.3 0.3 0.2 0.3
102 105 108 110 112
± ± ± ± ±
3 2 3 2 2
7.47 7.81 8.10 8.34
± ± ± ±
0.24 0.17 0.19 0.21
86.4 87.6 89.3 90.6
± ± ± ±
0.2 0.3 0.3 0.4
123 125 127 127
± ± ± ±
3 2 3 4
4.92 5.20 5.46 5.90
± ± ± ±
0.23 0.19 0.16 0.21
72.6 73.5 74.4 75.1
± ± ± ±
0.2 0.3 0.4 0.3
67 69 71 74
± ± ± ±
2 3 2 4
11.02 11.63 12.45 13.03
± ± ± ±
0.11 0.16 0.21 0.38
88.0 90.0 91.9 93.6
± ± ± ±
0.4 0.3 0.4 0.5
104 107 111 113
± ± ± ±
3 3 4 5
7.45 7.73 7.84 8.32
± ± ± ±
0.17 0.18 0.21 0.17
86.1 87.4 88.8 89.9
± ± ± ±
0.3 0.4 0.3 0.2
123 125 126 127
± ± ± ±
3 3 2 2
4.78 5.02 5.48 5.70
± ± ± ±
0.13 0.21 0.17 0.24
72.5 73.3 74.2 75.3
± ± ± ±
0.2 0.3 0.2 0.2
66 69 70 74
± ± ± ±
4 3 2 4
11.17 11.65 12.24 12.92
± ± ± ±
0.28 0.13 0.12 0.15
88.2 90.1 91.9 93.1
± ± ± ±
0.2 0.3 0.2 0.3
105 106 108 111
± ± ± ±
3 2 2 3
denatured state in the absence of the chemical denaturant (Fig. 4). Results given in Fig. 4A and B suggest a clear difference in the structural characteristics of the denatured states of Mb and barstar induced by heat and GdmCl. However, the structural characteristics of the GdmCl- and heat-induced denatured states of bcyt-c are, within experimental errors, identical (Fig. 4C). 3.3. Role of denatured state's structural properties on protein stabilization
Fig. 3. Heat-induced denaturation curves of proteins at pH 7.0. Denaturation profiles of Mb (A) and b-cyt-c (B) in the presence of different concentrations of GdmCl. Symbols (ο), (D), (7), (C), ( ) and (-) represent 0.4, 0.6, 0.8, 1.0, 1.25 and 1.5 M GdmCl. To maintain clarity all data points are not shown.
▫
denaturant gave the value of [q]l of the heat denatured protein in the absence of GdmCl. All values of [q]l at 0 M GdmCl were plotted as a function of wavelength to generate the spectrum of the heat-
Fig. 5 shows plots of DDGD versus [osmolyte]; DDGD is the difference between DGD values in the presence and absence of the osmolytes. These plots were analyzed for m-value (¼ dDDGD /d [osmolyte]) which measures sensitivity of DGD value of the protein on [osmolyte]. These m-values for each protein are given in Table 4. It is seen in this table (also see Fig. 5) that m-values of Mb and barstar are different for the heat- and GdmCl-denatured states. However, m-values of b-cyt-c for both denatured states are, within experimental errors, identical. Since m-value of a protein depends on the extent of exposure of protein groups on denaturation [19,44], we have estimated it theoretically using Eq. (5) for transfer of the protein from water to 1 M osmolyte solution. These m-values of proteins are compared with the experimentally measured values (see Fig. 6). We have observed that all osmolytes are stabilizing, and a good agreement was observed between predicted and experimental m-values in cases of sarcosine and TMAO (Fig. 6). However, the model fails to accurately predict the m-values of proteins in the case of glycine-betaine. It can be seen in Fig. 6 that the predicted values are underestimated. At present we do not know the source of this discrepancy. It should be noted that we have used myoglobin and bovine cytochrome c in our studies which have a heme prosthetic group. We have assumed that the effect of heme on total transfer-free energy of the proteins from water to 1 M osmolyte solution is negligible. It is interesting to recall an earlier study showing that the presence of heme group has negligible effect on the change in total ASA of protein on denaturation [45]. 4. Discussion 4.1. Heat- and GdmCl-induced denatured states Denatured states of proteins have been given great attention, especially since the structural characteristics of the denatured state
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Table 3 DCp values of proteins in the presence of different concentrations of osmolytes.
[Osmolytes], M Sarcosine 0.00 M 0.25 M 0.50 M 0.75 M 1.00 M TMAO 0.25 M 0.50 M 0.75 M 1.00 M GB 0.25 M 0.50 M 0.75 M 1.00 M
Mb
Barstar
b-cyt-c
DCp, kcal mol1 K1
DCp, kcal mol1 K1
DCp, kcal mol1 K1
2.49 2.44 2.40 2.43 2.38
± ± ± ± ±
0.06 0.05 0.06 0.07 0.05
1.28 1.24 1.26 1.24 1.27
± ± ± ± ±
0.04 0.07 0.09 0.05 0.07
1.24 1.22 1.23 1.19 1.17
± ± ± ± ±
0.05 0.03 0.06 0.07 0.08
2.43 2.40 2.37 2.31
± ± ± ±
0.03 0.03 0.05 0.04
1.25 1.21 1.25 1.26
± ± ± ±
0.08 0.06 0.08 0.05
1.22 1.21 1.23 1.19
± ± ± ±
0.04 0.06 0.07 0.09
2.44 2.42 2.40 2.34
± ± ± ±
0.05 0.06 0.07 0.05
1.26 1.30 1.21 1.32
± ± ± ±
0.09 0.05 0.04 0.07
1.24 1.21 1.15 1.18
± ± ± ±
0.04 0.06 0.07 0.09
appears to play a crucial role in many cellular processes [46e48]. Intensive studies have confirmed that for most proteins, the thermally denatured states are structurally non-identical to GdmClinduced denatured state [49e51]. Heat-induced denatured states of proteins, in fact, retain lots of residual structure that contains high amount of secondary and tertiary interactions [32,33]. Since residual structure plays an important role in many cellular processes [52], it will be interesting to investigate how cellular osmolytic compounds interact with denatured states containing residual structure and how this interaction is different from that with linear random coils. First of all, we characterized and compared heat-induced and GdmCl-induced denatured states of all the three proteins (barstar, Mb, b-cyt-c) using far-UV CD measurements. We observed that barstar and Mb retain a large amount of secondary structures in their heat-denatured states (Fig. 4A and B) as compared to the GdmCl-induced denatured state. Using the method given by Chen et al. [53], we determined a-helical content retained in the heatinduced denatured state of each protein and compared it with the native protein. We observed that the heat-induced denatured state of Mb retains 23.4% helical structure as compared to 71.8% helical content in the native state. Likewise, the helical content of barstar in the heat denatured state is 11.81% as compared to 41.45% in the native state. The results on barstar and Mb are in good agreements with the earlier reports that heat-induced denatured states of these proteins retain a large amount of secondary and tertiary structures [31,51]. Interestingly, for b-cyt-c, CD spectrum of the thermally denatured state is not significantly different from that of the GdmCl-denatured state (Fig. 4C). The helical content in the denatured b-cyt-c is almost zero (0.6%) as compared to that (34.0%) found in the native state. This finding is in excellent agreement with earlier results showing that GdmCl and heatinduced denatured states are identical in b-cyt-c [49,51]. We conclude that denatured states induced by heat and GdmCl are structurally dissimilar for both barstar and Mb, but is similar in the case of b-cyt-c. 4.2. Osmolyte-induced protein stabilization The thermodynamic basis of the osmolyte-induced protein folding/unfolding (or stabilization/destabilization) has been explained in terms of osmolyte's preferential exclusion from, or preferential binding to the protein surface [54], which is supported by recent observations on the transfer-free energy of the protein groups from the solvent water to the osmolyte aqueous solutions
[55]. Based on these findings, preferential exclusion stabilizes proteins by shifting the denaturation equilibrium, N state 4 D state, towards the left, while preferential binding destabilizes proteins by shifting the denaturation equilibrium toward the right, and therefore making balance of binding and exclusion to be a determining factor for the protein stabilization by an osmolyte. Thus, what effect osmolytes will have on the denaturation equilibrium, N state 4 D state under the native condition could only be understood by measuring DGD in different solvent conditions. The observed DGD values, summarized in Tables 1 and 2, and DDGD versus [osmolyte] plots (Fig. 5A and B), show that there is a clear difference in the extent of protein stabilization (DGD ) by an osmolyte obtained from heat- and GdmCl-induced denaturations of Mb and barstar. If this observed difference in the extent of protein stabilization from the two different modes of denaturation is indeed due to difference in the structural characteristics of the heat- and GdmCl-induced denatured states, then there should not be any difference in DDGD values obtained from GdmCl- and heatinduced denaturation studies in the case of b-cyt-c, for there exists no difference in the structural characteristics of the heat-denatured and GdmCl-denatured states ([49,51]; also see Fig. 4C). Fig. 5C shows that, for each osmolyte, this is indeed true for b-cyt-c, for the measured DDGD values obtained from GdmCl- and heat-induced denaturation measurements are, within experimental errors, identical. All these results strengthen our hypothesis that the variation in the structural characteristics of the denatured state results in the difference in the magnitude of protein stabilization in the presence of osmolytes or protein-osmolyte interaction. It is known from the transfer-free energy measurements that osmolytes interact unfavorably with the peptide backbone exposed upon unfolding (i.e., this interaction forces protein to fold) and favorably with side chains (i.e., this interaction opposes folding). Because GdmCl induces an apparently random coil structure, all the peptide backbones and side chains are fully exposed to the solvent, thereby allowing osmolytes to interact with all the protein groups. Since the number of the peptide group (eNHeCHeCOe) is very much larger than that of side chains interacting favorably with the osmolyte, the unfavorable interaction predominates over the favorable interactions, thereby potentiating the preferential hydration effect in case of random coil polypeptides. Persistence of local structures in the heat-denatured states results in burying of some of the peptide backbones which will be unexposed to the solvent. Consequently, osmolytes will interact with less exposed peptide backbone (leading to low preferential hydration) as compared to the fully exposed polypeptides, and therefore, the
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Fig. 4. Structural characteristics of heat- and GdmCl-induced denatured states of proteins. The far-UV CD spectra of Mb (A), barstar (B) and b-cyt-c (C) were corrected for solvent effect, if any (see text). Solid line, long dashes, and short dashes represent different states of proteins. Inset in the figure shows dependence of mean residual ellipticity at 222 nm on [GdmCl].
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Fig. 6. Experimental and predicted m-values of Mb, barstar and b-cyt-c in the presence of different osmolytes. Gray and black bar represent respectively predicted and experimental m-values of proteins in different osmolytes.
protein groups. These arguments support our observations shown in Fig. 5. This is noteworthy that the osmolyte effect on glutaminyltRNA synthetase from E. coli was shown to depend on the nature of the denatured states of the protein [56]. Fig. 5. DDGD versus [Methylamine] plots of Mb (A), barstar (B), and b-cyt-c (C) at pH 7.0. Curves 1 (TMAO), 2 (sarcosine) and 3 (GB) represent data obtained from GdmClinduced denaturation, and curves 4 (TMAO), 5 (sarcosine) and 6 (GB) represent data obtained from heat-induced denaturation.
magnitude of protein stabilization is less. This therefore suggests that the preferential hydration effects induced by osmolytes on the heat-induced and GdmCl-induced denatured states may not be identical due to possible variations in the exposed and/or buried
4.3. Correlation between m-values and Dai On the molecular level, the degree of unfolding of the heat- and GdmCl-denatured states are directly related to the fractional exposure of amino acid residues of the protein. Therefore, it is important to know how the fractional exposure (Dai) values of the heat- and GdmCl-induced denatured states are related to osmolyteinduced protein stability. The extent of Dai of each residue of a
Table 4 m-values estimated from the linear plots of DDGD versus osmolyte concentration. Mode of denturation
m-value, kcal mol1 M1
Myoglobin
N4D N4D N4D N4D N4D N4D
m-sarcosine
3.77 1.26 4.26 1.24 3.03 1.22
(GdmCl-induced) (Heat-induced) (GdmCl-induced) (Heat-induced) (GdmCl-induced) (Heat-induced)
m-TMAO m-GB
± ± ± ± ± ±
0.13 0.07 0.07 0.04 0.08 0.05
Barstar 2.38 1.40 2.56 1.35 2.32 1.15
± ± ± ± ± ±
0.14 0.06 0.06 0.09 0.16 0.11
b-cyt-c 2.61 2.37 2.80 2.40 2.51 2.32
± ± ± ± ± ±
0.11 0.24 0.15 0.15 0.08 0.14
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protein directly depends on the m-value (i.e., the sensitivity of the DGD of the protein on [osmolyte]). So, estimation of m-value would be an important parameter to correlate the extent of unfolding and the magnitude of stabilization of the protein. The m-value for a protein-co-solvent system can be calculated by the linear analysis of DDGD (co-solvent) versus [co-solvent] plot [19]. It is seen in Table 4 that there is 2e3 fold increase in m-values obtained from the GdmCl-induced denaturation as compared to their heatinduced ones for barstar and Mb. However, there is no significant difference in the observed m-values in the case of b-cyt-c. These observations clearly show that accessible surface area of the denatured state is the determining factor in osmolytes-induced protein stability. Furthermore, on the theoretical basis, Tanford's transfer-free energy model [39] is one method to predict and explain the thermodynamics of protein stability (forcing protein to fold or unfold) in osmolyte solution. The m-value which is a measure of an osmolyte's tendency to force protein to either fold or unfold, can be predicted theoretically using Tanford's transfer-energy model using Bolen's values for the transfer of amino acid residues from water to 1 M osmolyte solution [41,57]. These methods have been successfully employed to predict m-values of a wide range of monomeric, ligand free proteins for a number of osmolytic conditions, and an excellent agreement was found between predicted and experimental data [41,57,58]. To calculate m-value for any protein, first of all we need Dai (fractional change in accessible surface area of the ith type of residue), which can be obtained using Eq. (6). As it is seen in this equation, the evaluation of Da requires ASA of amino acid residues in the denatured state as well as in the native state. The native state solvent accessibility has been obtained from atomic coordinate of these proteins using DSSP server based program [40] but for denatured state solvent accessibility various models has been proposed. These models for the calculation of the denatured state solvent accessibility have been extensively reviewed by Ali et al. [59], and they suggest that the Creamer (LB) and Bernado (maximum) models gave an excellent prediction for m-values. In our case we have used Bernado's denatured state model to calculate theoretical m-values. We have used this model because this model shows very small influence of both the protein size and protein amino acid composition on average ASA of individual residues [42]. Furthermore, Bernado's values for the upper and lower limits can serve to bracket the expected solvent accessibility for the denatured state. It is seen in Fig. 6 that (a) predicted m-values obtained using Bernado's denatured state model, for all three proteins are in agreement with the experimental m-values for two osmolytes sarcosine and TMAO, and (b) in case of glycine-betaine the predicted values were underestimated. At the moment, we ignore the exact reason for this discrepancy, but similar differences in predicted and experimental m-values have been observed in some earlier studies on proteins [60,61].
5. Conclusions We conclude that the extent of protein stabilization by osmolytes, obtained from GdmCl- and heat-induced denaturation measurements, is different due to differences in the structural characteristics of the denatured states induced by these two different denaturants. Caution must, therefore, be taken in comparing an experimental m-value of the protein with the theoretical m-value obtained using Eqs. (5) and (6), for it is possible only when amino acid residues are fully exposed to the solvent on denaturation.
Acknowledgments We are grateful to Dr. J. B. Udgaonkar (National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore) for his help in the preparation of barstar. FA is thankful to Department of Science and Technology, India (SB/SO/BB-71/ 2010(G)) for financial support. We sincerely thank Department of Science and Technology, India for FIST support [SR/FST/LS1-541/ 2012]. Footnote: ASA (accessible surface area) is a way of quantifying burial of amino acid residues in a protein, and it is the extent to which atoms on the surface of a protein can form contacts with the solvent water. References [1] P.H. Yancey, Biologist 50 (2003) 126e131. [2] P.H. Yancey, Sci. Prog. 87 (2004) 1e24. [3] P.H. Yancey, M.E. Clark, S.C. Hand, R.D. Bowlus, G.N. Somero, Science 217 (1982) 1214e1222. [4] N. Gregersen, P. Bross, S. Vang, J.H. Christensen, Annu. Rev. Genom. Hum. Genet. 7 (2006) 103e124. [5] F. Chiti, C.M. Dobson, Annu. Rev. Biochem. 75 (2006) 333e366. [6] P.H. Yancey, J. Exp. Biol. 208 (2005) 2819e2830. [7] S. Jamal, N.K. Poddar, L.R. Singh, T.A. Dar, V. Rishi, F. Ahmad, FEBS J. 276 (2009) 6024e6032. [8] P.H. Yancey. In: G.N. Somero, C.B. Osmond, C.L. Bolis (Eds), Springer-Verlag, Berlin, 1992. p. 19. [9] L.R. Singh, N.K. Poddar, T.A. Dar, S. Rahman, R. Kumar, F. Ahmad, J. Iran. Chem. Soc. 8 (2011) 1e23. [10] R.E. MacMillen, A.K. Lee, Science 158 (1967) 383e385. [11] P.H. Yancey, G.N. Somero, J. Exp. Zool. 212 (1980) 205e213. [12] J. Seeliger, K. Estel, N. Erwin, R. Winter, Phys. Chem. Chem. Phys. 15 (2013) 8902e8907. [13] K. Hatori, T. Iwasaki, R. Wada, Biophys. Chem. 193e194 (2014) 20e26. [14] I. Baskakov, A. Wang, D.W. Bolen, Biophys. J. 74 (1998) 2666e2673. [15] T.Y. Lin, S.N. Timasheff, Biochemistry 33 (1994) 12695e12701. [16] S. Taneja, F. Ahmad, Biochem. J. 303 (Pt 1) (1994) 147e153. [17] I. Haque, A. Islam, R. Singh, A.A. Moosavi-Movahedi, F. Ahmad, Biophys. Chem. 119 (2006) 224e233. [18] I. Haque, R. Singh, A.A. Moosavi-Movahedi, F. Ahmad, Biophys. Chem. 117 (2005) 1e12. [19] L.R. Singh, T.A. Dar, I. Haque, F. Anjum, A.A. Moosavi-Movahedi, F. Ahmad, Bba Proteins Proteom. 1774 (2007) 1555e1562. [20] L.R. Singh, T.A. Dar, S. Rahman, S. Jamal, F. Ahmad, Biochim. Biophys. Acta 1794 (2009) 929e935. [21] R. Singh, I. Haque, F. Ahmad, J. Biol. Chem. 280 (2005) 11035e11042. [22] A.J. Wang, D.W. Bolen, Biophys. J. 71 (1996) 2117e2122. [23] I. Haque, R. Singh, F. Ahmad, A.A. Moosavi-Movahedi, FEBS Lett. 579 (2005) 3891e3898. [24] S. Khan, Z. Bano, L.R. Singh, M.I. Hassan, A. Islam, F. Ahmad, PLoS One 8 (2013) e72533. [25] T. Arakawa, S.N. Timasheff, Methods Enzymol. 114 (1985) 49e77. [26] S.N. Timasheff, Annu. Rev. Biophys. Biomol. Struct. 22 (1993) 67e97. [27] S.N. Timasheff, Biochemistry 41 (2002) 13473e13482. [28] P. Bruzdziak, B. Adamczak, E. Kaczkowska, J. Czub, J. Stangret, Phys. Chem. Chem. Phys. 17 (2015) 23155e23164. [29] D.W. Bolen, I.V. Baskakov, J. Mol. Biol. 310 (2001) 955e963. [30] Y. Liu, D.W. Bolen, Biochemistry 34 (1995) 12884e12891. [31] R. Khurana, J.B. Udgaonkar, Biochemistry 33 (1994) 106e115. [32] C. Tanford, Adv. Protein Chem. 23 (1968) 121e282. [33] F. Franks, Adv. Protein Chem. 46 (1995) 105e139. [34] D. Puett, J. Biol. Chem. 248 (1973) 4623e4634. [35] E. Margoliash, N. Frohwirt, Biochem. J. 71 (1959) 570e572. [36] M.M. Santoro, D.W. Bolen, Biochemistry 27 (1988) 8063e8068. [37] S. Yadav, F. Ahmad, Anal. Biochem. 283 (2000) 207e213. [38] A. Sinha, S. Yadav, R. Ahmad, F. Ahmad, Biochem. J. 345 (Pt 3) (2000) 711e717. [39] C. Tanford, Adv. Protein Chem. 24 (1970) 1e95. [40] W. Kabsch, C. Sander, Biopolymers 22 (1983) 2577e2637. [41] M. Auton, D.W. Bolen, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 15065e15068. [42] P. Bernado, M. Blackledge, J. Sancho, Biophys. J. 91 (2006) 4536e4543. [43] W.J. Becktel, J.A. Schellman, Biopolymers 26 (1987) 1859e1877. [44] F. Ahmad, C.C. Bigelow, Biopolymers 25 (1986) 1623e1633. [45] C. Ganesh, N. Eswar, S. Srivastava, C. Ramakrishnan, R. Varadarajan, FEBS Lett. 454 (1999) 31e36. [46] A. Mohan, C.J. Oldfield, P. Radivojac, V. Vacic, M.S. Cortese, A.K. Dunker, V.N. Uversky, J. Mol. Biol. 362 (2006) 1043e1059. [47] A.P. Capaldi, C. Kleanthous, S.E. Radford, Nat. Struct. Biol. 9 (2002) 209e216. [48] K. Ding, J.M. Louis, A.M. Gronenborn, J. Mol. Biol. 335 (2004) 1299e1307. [49] Y. Hagihara, M. Hoshino, D. Hamada, M. Kataoka, Y. Goto, Fold. Des. 3 (1998)
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Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins Safikur Rahman 1, Syed Ausaf Ali, Asimul Islam, Md. Imtaiyaz Hassan, Faizan Ahmad* Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 August 2015 Received in revised form 4 November 2015 Accepted 21 November 2015 Available online 12 December 2015
Here we have generated two different denatured states using heat- and guanidinium chloride (GdmCl) induced denaturations of three disulfide bond free proteins (barstar, cytochrome-c and myoglobin). We have observed that these two denatured states of barstar and myoglobin are structurally and energetically different, for, heat-induced denatured state contains many un-melted residual structure that has a significant amount of secondary and tertiary interactions. We show that structural properties of the denatured state determine the magnitude of the protein stabilization in terms of Gibbs free energy change (DGD ) induced by an osmolyte, i.e., the greater the exposed surface area, the greater is the stabilization. Furthermore, we predicted the m-values (ability of osmolyte to fold or unfold proteins) using Tanford's transfer-free energy model for the transfer of proteins to osmolyte solutions. We observed that, for each protein, m-value is comparable with our experimental data in cases of TMAO (trimethylamine-N-oxide) and sarcosine. However, a significant discrepancy between predicted and experimental m-values were observed in the case of glycine-betaine. © 2015 Elsevier Inc. All rights reserved.
Keywords: Osmolytes Methylamines Protein stabilization Accessible surface area Gibbs free energy
1. Introduction Many proteins refold from their unfolded state to attain the native functional state. The correct folding of the protein polypeptide chain is determined not only by its amino acid sequence but also by the solvent environment. Cells are quite often exposed to widely fluctuating environmental conditions such as extremes of temperature, pH, cellular dehydration, desiccation, high extracellular salts, and even the presence of denaturing concentrations of urea inside the cell [1e3]. Protein folding in the cell is quite often challenged because of these environmental stresses. Failure to circumvent these hostile external environmental stresses may lead to various proteopathies, including protein misfolding, aggregation, amyloid formation and enhanced proteosomal degradation due to protein destabilization and eventually apoptosis [4,5]. Even the protein quality control system, including molecular chaperones, specialized intracellular proteases and accessory factors that
* Corresponding author. E-mail address: [email protected] (F. Ahmad). 1 Present address: Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India. http://dx.doi.org/10.1016/j.abb.2015.11.035 0003-9861/© 2015 Elsevier Inc. All rights reserved.
regulate the activity of chaperones and proteases, may also get damaged due to the extreme stresses. Interestingly, organisms or cells have a developed mechanism of accumulation of low molecular mass organic molecules called osmolytes which protect them against adverse environmental stresses. Osmolytes can be grouped into three chemical classes as polyols and sugars (e.g., trehalose, glycerol, inositols, etc.), amino acids (e.g., glycine, proline, etc.) and their derivatives (e.g., balanine, taurine, etc.) and combinations of methylamines (e.g., sarcosine, trimethylamine-N-oxide (TMAO), etc.) and urea [3,6]. Often, they are classified as compatible or counteracting based on their effects on both the stability and function of proteins [7]. Compatible osmolytes increase stability of proteins against thermal denaturation with little or no effect on their function near room temperature [8,9]. Representatives of this class include certain amino acids and polyols [8,9]. Counteracting osmolytes, on the other hand, are believed to have a special ability to protect intracellular proteins against inactivation and/or destabilization by urea [10e13]. That is, they cause changes that are opposite to the effect of urea on protein stability and function of the protein [1,9,14,15]. Organs like mammalian kidney and many animals like cartilaginous fishes and coelacanth that are rich in urea, employ these
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counteracting osmolytes including glycine-betaine, glycerophosphorylcholine (GPC) and TMAO [6,8,10]. There are two defining characteristics of compatible osmolytes, namely, they stabilize proteins against denaturation by heat or chemicals [16e21], and their presence in the cell does not largely alter protein functional activity [3,9,18,22e24]. The mechanism of osmolytes-induced protein stability has been studied extensively [3,9,25e28]. It has been widely accepted that osmolytes stabilize or induce protein folding due to preferential hydration effect [26,27]. Bolen et al. [29,30] from their elegant series of experiments that measured the transfer-free energy of the amino acid side chains and backbone, showed that the preferential hydration effect on proteins is chiefly because of the unfavorable interactions of osmolytes with the peptide backbone in the denatured state while the total contribution of amino acid side chains is little. Their studies led to the conclusion that osmolytes-induced thermodynamic stability of proteins is due to shift in denaturation equilibrium, N (native state) 4 D (denatured state), from right to the left. Although, osmolytes-facilitated protein stability has been deeply studied, most of these studies have been carried out on proteins having disulfide bond cross links or intrinsically unstructured proteins. Furthermore, to the best of our knowledge, no earlier studies compared osmolyte-induced thermodynamic stability of a protein obtained from heat- and GdmCl-induced denaturation, as it is widely known that these two modes of protein denaturation may give structurally two different denatured states [31e33]. To mimic the intercellular state of disulfide bonds containing proteins, we have used three different disulfide free proteins. We report that, for such a protein, the osmolyte-induced stabilizing effect in terms of Gibbs free energy change (DGD ), obtained from the GdmCl-induced denaturation is larger than that obtained from the heat-induced denaturation. Furthermore, we have theoretically predicted the m-value (ability of osmolyte to refold or unfold protein) and found a good correlation between theoretical and experimental m-values of all GdmCl-denatured proteins.
2. Materials and methods 2.1. Materials Commercial lyophilized preparation of horse heart myoglobin (Mb) and bovine cytochrome-c (b-cyt-c) were purchased from Sigma Chemical Co (St. Louis, MO). Barstar (from Bacillus amyloliquefaciens) was expressed in Escherichia coli and purified by the method published by Khurana and Udgaonkar [31]. GdmCl was purchased from MP Biomedical Inc. TMAO, Sarcosine and Glycinebetaine (GB) were obtained from Sigma Chemical Co (St. Louis, MO). These and other chemicals, which were of analytical grade, were used without further purification.
2.2. Preparation of protein solutions Mb and b-cyt-c were oxidized by dissolving them in 0.1 and 0.01% potassium ferricyanide solutions, respectively. These solutions were then dialyzed against several changes of 0.1 M KCl solution (pH 7.0) at 4 C. Barstar was also dialyzed in cold against 0.1 M KCl at pH 7.0. The concentrations of stock solutions of proteins were determined using molar absorption coefficient, ε (M1cm1) values of 171,000 at 409 nm for Mb [34], 106,000 at 409 nm for bcyt-c [35] and 23,000 at 280 nm for barstar [31]. All solutions for optical measurements were prepared in the desired degassed buffer containing 0.1 M KCl.
2.3. Spectroscopic determination of free energy change associated with GdmCl- and heat-induced denaturations GdmCl-induced denaturations of b-cyt-c, Mb and barstar in the absence and presence of different concentrations of each methylamine were monitored by following changes in [q]222 (molar ellipticity at 222 nm) at pH 7.0 and 25 C. The unfolding transitions of these proteins were reversible in the entire [g] (molar GdmCl concentration) range in the presence of each co-solute. Using a non-linear least-squares method, the entire data (y(g), [g]) of each denaturant-induced transition curve were analyzed for DGD , mg and Cm using the relation [36],
yðgÞ ¼
0 yN ðgÞ þ yD ðgÞ e½ðDGD þmg ½gÞ=RT 0 1 þ e½ðDGD þmg ½gÞ=RT
(1)
where y(g) is the observed optical property at [g], yN and yD are optical properties of the native and denatured protein molecules under the same experimental conditions in which y(g) was measured, DGD is the value of Gibbs free energy change (DGD) in the absence of denaturant, mg is the slope (vDGD/v[g])T,P, R is the universal gas constant, and T is the temperature in Kelvin. It should, however, be noted that the analysis of each GdmCl-induced transition curve was done assuming that unfolding is a two-state process and [g]-dependencies of yN(g) and yD(g) are linear (i.e., yN(g) ¼ aN þ bN [g] and yD(g) ¼ aD þ bD [g], where a and b are [g]independent parameters, and subscripts N and D represent these parameters for the native and denatured protein molecules, respectively). Heat-induce-denaturation studies were carried out in Jasco spectropolarimeter, Model J-1500-150 (JASCO Corporation, Japan), equipped with Peltier-type temperature controller, at a heating rate of 1 C/min. This scan rate was found to provide adequate time for equilibration. The change in secondary structure of the protein with increasing temperature was followed by measuring the far-UV CD at 222 nm. About 650 data points (data point at 0.1 C interval) of each transition curve were collected. After denaturation, the sample was immediately cooled down to measure reversibility of the process. Each heat-induced transition curve was analyzed for Tm and DHm using a non-linear least-squares analysis according to the relation:
yðTÞ ¼
yN ðTÞ þ yD ðTÞ exp ½ DHm =Rð1=T 1=Tm Þ 1 þ exp½ DHm =Rð1=T 1=Tm Þ
(2)
where y(T) is the optical property at temperature T (Kelvin), yN(T) and yD(T) are the optical properties of the native and denatured protein molecules at temperature T (Kelvin), and R is the gas constant. In the analysis of each heat-induced 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) ¼ aN þ bNT þ cNT2 and yD(T) ¼ aD þ bDT þ cDT2, where aN, bN, cN, aD, bD, and cD are temperature-independent coefficients) [37,38]. The value of the temperature-independent constant-pressure heat capacity change (DCp) was determined from the slope of the linear plots of DHm versus Tm, using the relation:
DCp ¼
dDHm dTm
(3) P
Using values of Tm, DHm and DCp, the value of DGD at any temperature T, DGD(T) was estimated using the GibbseHelmholtz equation:
S. Rahman et al. / Archives of Biochemistry and Biophysics 591 (2016) 7e17
DGD ðTÞ ¼ DHm
Tm T Tm
T DCp ðTm TÞ þ T ln Tm
(4)
2.4. Estimation of m-values using Tanford's transfer-free energy model [39] The program DSSP [40] was used to estimate accessible surface area (ASA) of proteins using PDB coordinates of 1YMB (Mb from horse heart), 1BTA (barstar from Bacillus amyloliquefaciens) and 2B4Z (b-cyt-c from bovine heart). Transfer-free energy values from water to 1.0 M osmolyte solution for side chains and peptide
9
backbone were used as given by Auton and Bolen [41] who showed that the total transfer-free energy of a protein is related to mos (ability of osmolyte to unfold or re-fold the protein) through the relation:
mos ¼
X
ni Dai Dgtr;i
(5)
where ni is the number of the ith type of residue (eNHeCHReCOe) in the protein, Dgtr,i is the transfer-free energy change of the ith type of residue when it is transferred from water to the 1.0 M osmolyte solution, and Dai is the fractional increase in the exposure of the ith type of residue on denaturation. Dai was calculated using the relation:
Fig. 1. GdmCl-induced denaturation curves of proteins in the presence and absence of osmolytes at pH 7.0 and 25 C. GdmCl-induced denaturation profiles of Mb (A), barstar (B) and b-cyt-c (C) in the absence and presence of methylamines. Control (ο) represents denaturation curve in the absence of osmolytes. Symbols (D), (7) and ( ) represent 0.5 M sarcosine, 0.5 M TMAO and 0.5 M GB, respectively, while (:), (;) and (-) represent1.0 M TMAO, 1.0 M sarcosine and 1.0 M GB, respectively. Far-UV CD spectra of the native (solid line), GdmCl denatured in 3.0 M GdmCl (line-dot) and renatured Mb (D), barstar (E) and b-cyt-c (F) in 1.0 M GdmCl (long dash).
▫
10
Dai ¼
S. Rahman et al. / Archives of Biochemistry and Biophysics 591 (2016) 7e17
P XASA;i FASA;i XASA;i
(6)
XASA,i and FASA,i are accessible surface areas of the ith type of residue in the denatured and native states, respectively. Using DSSP server and PDB coordinates, we calculated FASA,i value of each type of residue in the native state. For estimation of XASA,i we have used denatured state model of Bernado et al. [42]. 3. Results 3.1. Protein stability from heat and GdmCl-induced denaturation studies To investigate the interaction of osmolyte with heat-induced and GdmCl-induced denatured states, we have intentionally chosen three proteins (Mb, barstar and b-cyt-c) which do not contain disulfide bond. To estimate the effect of osmolytes on DGD (at 25 C) of these proteins, we measured heat-induced and GdmClinduced denaturation curves in the presence of various concentrations (0, 0.25, 0.50, 0.75, and 1.0 M) of each of the methylamines (sarcosine, TMAO and GB) by following changes in [q]222 (a probe for change in secondary structure). Denaturation (heat- or GdmClinduced) of each of these proteins was reversible in the entire range of [osmolyte], the molar concentration of the methylamine. Fig. 1 shows GdmCl-induced denaturation curves of Mb, barstar and bcyt-c in the presence of 0, 0.5 and 1.0 M osmolytes at pH 7.0. Results at other concentrations of each osmolyte are shown elsewhere (see Figure 1 in ref. [#]). It was observed that for each protein the dependence of yN and yD on [g], the molar concentration of GdmCl, is independent of the osmolyte type and its concentration (see Fig. 1 and (Fig. 1 in ref. [#])). Furthermore, it was observed that, for each protein, yN does not depend on [g]. However, yD of Mb and bcyt-c showed slight dependence on [g]. Each transition curve, which was measured at least three times, was analyzed for thermodynamic parameters using Eq. (1). Values of DGD , mg and Cm thus obtained are given in Table 1. Fig. 2 shows heat-induced denaturation curves of Mb, barstar and b-cyt-c in the presence of 0, 0.5 and 1.0 M sarcosine, TMAO and GB at pH 7.0. Fig. 2 in ref. [#] shows denaturation curves at other concentrations of each osmolytes at pH 7.0. Figs. 3e5 in the ref. [#] show denaturation curves of these proteins in the presence of 0,
0.25, 0.50 and 1.0 M osmolyte at pH values other than 7.0. Each transition curve of a protein at a given [osmolyte] and pH was analyzed for thermodynamic parameters (Tm and DHm) using a non-linear least-squares method that involves fitting the entire ([q]222, T) data of the transition curve to Eq. (2) with all eight free parameters (aN, bN, cN, aD, bD, cN, Tm and DHm). Values of Tm and DHm of all three proteins in the presence and absence of different osmolytes at pH 7.0 are given in Table 2. The values of these parameters of proteins in the absence and presence of different concentrations of each osmolyte at pH values other than pH 7.0 are given in Tables 1e3 in ref. ([#]). It may be noted that for heatinduced denaturations, we were not able to obtain complete transition curves in cases of Mb and b-cyt-c. So in order to bring down the thermal transition curves in the measurable range, we carried out denaturation studies in the presence of 0.6 and 1.25 M GdmCl for Mb and b-cyt-c, respectively. To correct for the contribution of GdmCl to the observed Tm and DHm, we carried out measurements of denaturation curves of these proteins in the presence of three more GdmCl concentrations (0.4, 0.8 and 1.0 M for Mb; and 0.6, 1.0 and 1.5 M for b-cyt-c) at different pH values. The results of such measurements of pH 7.0 are shown in Fig. 3. Corrections were done based on the method published earlier [21]. These corrected values of Tm and DHm are entered in Table 2 and Tables 1 and 2 in Ref. [Ref. #]. To estimate DCp of a protein at a given [osmolyte], DHm values were determined at number of Tm values by varying the pH, and the slope of the straight line of the DHm versus Tm plot is used to estimate DCp using Eq. (3) [43]. Values of DHm and Tm at different osmolyte concentrations and pH values are given in Table 2 and Tables 1e3 in ref. [#]. It should be noted that for a protein at a given [osmolyte], all 15 (DHm, Tm) values obtained from triplicate measurements at all pH values were used to construct the DHm versus Tm plot to determine DCp. Table 3 shows values of DCp of all proteins in the presence of different concentrations of sarcosine, TMAO and GB. It is seen in this table that DCp of proteins does not depend on [osmolyte]. At a constant pH and [osmolyte], DGD , the value of DGD in the absence of GdmCl at 25 C, was estimated using Eq. (4) with known values of DHm, Tm and DCp. However, this estimation requires a large extrapolation. Hence, a large error may be associated with DGD determination due to errors in the estimations of DHm, Tm, and DCp. We have used Becktel and Schellman's procedure [43] to
Table 1 Thermodynamic parameters associated with the GdmCl-induced denaturation of Mb, barstar and b-cyt-c in the absence and presence of different concentrations of osmolytes at pH 7.0 and 25 C. [Osmolytes] M
Sarcosine 0.00 0.25 0.50 0.75 1.00 TMAO 0.25 0.50 0.75 1.00 GB 0.25 0.50 0.75 1.00
Mb
Barstar
b-cyt-c
DGD
mg
Cm
DGD
mg
Cm
DGD
mg
Cm
kcal mol1
kcal mol1 M1
M
kcal mol1
Kcal mol1 M1
M
kcal mol1
kcal mol1 M1
M
7.08 8.01 8.98 10.01 10.85
± ± ± ± ±
0.26 0.25 0.24 0.22 0.21
4.43 4.42 4.51 4.47 4.49
± ± ± ± ±
0.10 0.07 0.09 0.10 0.12
1.60 1.81 1.99 2.23 2.42
± ± ± ± ±
0.06 0.09 0.06 0.09 0.09
4.98 5.48 5.89 6.61 7.35
± ± ± ± ±
0.21 0.16 0.21 0.18 0.20
2.63 2.62 2.58 2.53 2.59
± ± ± ± ±
0.09 0.04 0.05 0.10 0.08
1.89 2.09 2.28 2.61 2.83
± ± ± ± ±
0.11 0.06 0.09 0.08 0.08
10.60 11.33 12.02 12.78 13.20
± ± ± ± ±
0.21 0.25 0.27 0.28 0.24
3.99 4.03 4.04 4.06 3.98
± ± ± ± ±
0.06 0.0.8 0.10 0.07 0.04
2.66 2.81 2.97 3.15 3.31
± ± ± ± ±
0.15 0.12 0.14 0.13 0.12
8.09 9.04 10.29 11.34
± ± ± ±
0.23 0.17 0.22 0.29
4.39 4.36 4.50 4.51
± ± ± ±
0.07 0.08 0.04 0.07
1.85 2.08 2.29 2.52
± ± ± ±
0.10 0.11 0.06 0.08
5.53 6.14 6.74 7.51
± ± ± ±
0.17 0.24 0.13 0.21
2.68 2.76 2.56 2.65
± ± ± ±
0.08 0.09 0.07 0.08
2.06 2.22 2.63 2.83
± ± ± ±
0.09 0.07 0.07 0.09
11.27 12.31 12.98 13.39
± ± ± ±
0.28 0.25 0.24 0.29
4.01 4.17 4.06 3.97
± ± ± ±
0.10 0.08 0.09 0.10
2.81 2.95 3.19 3.37
± ± ± ±
0.14 0.12 0.15 0.10
7.95 8.42 9.39 10.14
± ± ± ±
0.19 0.21 0.27 0.25
4.42 4.52 4.55 4.44
± ± ± ±
0.09 0.07 0.06 0.08
1.80 1.86 2.06 2.28
± ± ± ±
0.09 0.05 0.07 0.08
5.54 5.94 6.72 7.24
± ± ± ±
0.17 0.24 0.18 0.21
2.54 2.55 2.69 2.68
± ± ± ±
0.04 0.08 0.07 0.05
2.18 2.33 2.50 2.70
± ± ± ±
0.10 0.07 0.14 0.06
11.23 12.03 12.53 12.92
± ± ± ±
0.19 0.29 0.35 0.15
4.04 4.02 3.89 3.85
± ± ± ±
0.09 0.10 0.11 0.05
2.78 2.99 3.22 3.35
± ± ± ±
0.12 0.09 0.10 0.12
S. Rahman et al. / Archives of Biochemistry and Biophysics 591 (2016) 7e17
11
Fig. 2. Heat-induced denaturation curves of proteins at pH 7.0. Denaturation profiles of Mb (A), barstar (B) and b-cyt-c (C) in the absence and presence of methylamines. Denaturation curves in cases of Mb and b-cyt-c were obtained in the presence of 0.6 and 1.25 M GdmCl, respectively. Symbols have same meaning as in Fig. 1. To maintain clarity all data points are not shown. Far-UV CD spectra of the native (solid line), heat-denatured at 85 C (line-dot) and renatured Mb (D), barstar (E) and b-cyt-c (F) at 20 C (long dash).
determine the maximum and minimum errors associated with the DGD determination in a given solvent condition. As there were three independent measurements of DHm and Tm of a protein at the given pH and [osmolyte], we obtained six values of DGD (three maximum and three minimum values). All of these six values were used to determine the average DGD and the mean error. The average values of DGD of all proteins at pH 7.0 are given in Table 2. Tables 1e3 in ref. [#] show the average values at other pH values. 3.2. Far-UV CD properties of denatured states Is there a structural difference between the GdmCl- and heatinduced denatured states of a protein? To answer this question, the far-UV CD (measure of change in secondary structure) measurements were done for all proteins in the presence of 4.0 M
GdmCl at 25 C and at 85 C (see Fig. 4). It may, however, be noted that the heat-denatured states of b-cyt-c and Mb obtained here already contained 1.25 M and 0.6 M GdmCl respectively, as heatinduced denaturation in the absence of the chemical denaturant is not complete at 85 C. Therefore, it is important to know whether the chemical denaturant has any effect on the heat-denatured state of these two proteins. For this, the far-UV CD spectra of the denatured b-cyt-c and Mb in the presence of different concentrations of GdmCl (0.4, 0.6, 0.8 and 1.0 for Mb and 0.6, 1.0, 1.25, 1.5 for b-cyt-c) were measured at 85 C (see Fig. 6 in [ref. #]). Values of [q] at different wavelengths were read from each spectrum measured in a given concentration of the chemical denaturant. Values of [q] at each wavelength were plotted as a function of [GdmCl] (e.g., see Fig. 4, inset). It was observed that the plot of [q]l versus [GdmCl] is linear, and a linear extrapolation of this plot to 0 M of the chemical
12
S. Rahman et al. / Archives of Biochemistry and Biophysics 591 (2016) 7e17
Table 2 Thermodynamic parameters associated with the thermal denturation of Mb, barstar and b-cyt-c in the absence and presence of different concentrations of osmolytes at pH 7.0. [Osmolytes] M
Mb
Barstar
DGD kcal mol Sarcosine 0.00 0.25 0.50 0.75 1.00 TMAO 0.25 0.50 0.75 1.00 GB 0.25 0.50 0.75 1.00
1
C
b-cyt-c
DGD
DHm
Tm
kcal mol
1
kcal mol
1
DGD
DHm
Tm C
kcal mol
1
kcal mol
Tm
D Hm
1
kcal mol1
C
7.10 7.41 7.75 7.99 8.35
± ± ± ± ±
0.19 0.16 0.34 0.35 0.21
85.5 86.3 88.2 90.4 91.9
± ± ± ± ±
0.3 0.3 0.4 0.2 0.3
121 122 124 126 127
± ± ± ± ±
2 2 3 3 3
4.55 4.82 5.27 5.59 5.95
± ± ± ± ±
0.23 0.19 0.17 0.22 0.21
72.0 72.4 73.3 74.7 75.8
± ± ± ± ±
0.3 0.1 0.2 0.2 0.4
65 66 70 72 74
± ± ± ± ±
2 3 3 4 3
10.60 11.24 11.72 12.42 12.98
± ± ± ± ±
0.23 0.28 0.16 0.27 0.21
86.6 88.5 90.0 92.3 93.8
± ± ± ± ±
0.4 0.3 0.3 0.2 0.3
102 105 108 110 112
± ± ± ± ±
3 2 3 2 2
7.47 7.81 8.10 8.34
± ± ± ±
0.24 0.17 0.19 0.21
86.4 87.6 89.3 90.6
± ± ± ±
0.2 0.3 0.3 0.4
123 125 127 127
± ± ± ±
3 2 3 4
4.92 5.20 5.46 5.90
± ± ± ±
0.23 0.19 0.16 0.21
72.6 73.5 74.4 75.1
± ± ± ±
0.2 0.3 0.4 0.3
67 69 71 74
± ± ± ±
2 3 2 4
11.02 11.63 12.45 13.03
± ± ± ±
0.11 0.16 0.21 0.38
88.0 90.0 91.9 93.6
± ± ± ±
0.4 0.3 0.4 0.5
104 107 111 113
± ± ± ±
3 3 4 5
7.45 7.73 7.84 8.32
± ± ± ±
0.17 0.18 0.21 0.17
86.1 87.4 88.8 89.9
± ± ± ±
0.3 0.4 0.3 0.2
123 125 126 127
± ± ± ±
3 3 2 2
4.78 5.02 5.48 5.70
± ± ± ±
0.13 0.21 0.17 0.24
72.5 73.3 74.2 75.3
± ± ± ±
0.2 0.3 0.2 0.2
66 69 70 74
± ± ± ±
4 3 2 4
11.17 11.65 12.24 12.92
± ± ± ±
0.28 0.13 0.12 0.15
88.2 90.1 91.9 93.1
± ± ± ±
0.2 0.3 0.2 0.3
105 106 108 111
± ± ± ±
3 2 2 3
denatured state in the absence of the chemical denaturant (Fig. 4). Results given in Fig. 4A and B suggest a clear difference in the structural characteristics of the denatured states of Mb and barstar induced by heat and GdmCl. However, the structural characteristics of the GdmCl- and heat-induced denatured states of bcyt-c are, within experimental errors, identical (Fig. 4C). 3.3. Role of denatured state's structural properties on protein stabilization
Fig. 3. Heat-induced denaturation curves of proteins at pH 7.0. Denaturation profiles of Mb (A) and b-cyt-c (B) in the presence of different concentrations of GdmCl. Symbols (ο), (D), (7), (C), ( ) and (-) represent 0.4, 0.6, 0.8, 1.0, 1.25 and 1.5 M GdmCl. To maintain clarity all data points are not shown.
▫
denaturant gave the value of [q]l of the heat denatured protein in the absence of GdmCl. All values of [q]l at 0 M GdmCl were plotted as a function of wavelength to generate the spectrum of the heat-
Fig. 5 shows plots of DDGD versus [osmolyte]; DDGD is the difference between DGD values in the presence and absence of the osmolytes. These plots were analyzed for m-value (¼ dDDGD /d [osmolyte]) which measures sensitivity of DGD value of the protein on [osmolyte]. These m-values for each protein are given in Table 4. It is seen in this table (also see Fig. 5) that m-values of Mb and barstar are different for the heat- and GdmCl-denatured states. However, m-values of b-cyt-c for both denatured states are, within experimental errors, identical. Since m-value of a protein depends on the extent of exposure of protein groups on denaturation [19,44], we have estimated it theoretically using Eq. (5) for transfer of the protein from water to 1 M osmolyte solution. These m-values of proteins are compared with the experimentally measured values (see Fig. 6). We have observed that all osmolytes are stabilizing, and a good agreement was observed between predicted and experimental m-values in cases of sarcosine and TMAO (Fig. 6). However, the model fails to accurately predict the m-values of proteins in the case of glycine-betaine. It can be seen in Fig. 6 that the predicted values are underestimated. At present we do not know the source of this discrepancy. It should be noted that we have used myoglobin and bovine cytochrome c in our studies which have a heme prosthetic group. We have assumed that the effect of heme on total transfer-free energy of the proteins from water to 1 M osmolyte solution is negligible. It is interesting to recall an earlier study showing that the presence of heme group has negligible effect on the change in total ASA of protein on denaturation [45]. 4. Discussion 4.1. Heat- and GdmCl-induced denatured states Denatured states of proteins have been given great attention, especially since the structural characteristics of the denatured state
S. Rahman et al. / Archives of Biochemistry and Biophysics 591 (2016) 7e17
13
Table 3 DCp values of proteins in the presence of different concentrations of osmolytes.
[Osmolytes], M Sarcosine 0.00 M 0.25 M 0.50 M 0.75 M 1.00 M TMAO 0.25 M 0.50 M 0.75 M 1.00 M GB 0.25 M 0.50 M 0.75 M 1.00 M
Mb
Barstar
b-cyt-c
DCp, kcal mol1 K1
DCp, kcal mol1 K1
DCp, kcal mol1 K1
2.49 2.44 2.40 2.43 2.38
± ± ± ± ±
0.06 0.05 0.06 0.07 0.05
1.28 1.24 1.26 1.24 1.27
± ± ± ± ±
0.04 0.07 0.09 0.05 0.07
1.24 1.22 1.23 1.19 1.17
± ± ± ± ±
0.05 0.03 0.06 0.07 0.08
2.43 2.40 2.37 2.31
± ± ± ±
0.03 0.03 0.05 0.04
1.25 1.21 1.25 1.26
± ± ± ±
0.08 0.06 0.08 0.05
1.22 1.21 1.23 1.19
± ± ± ±
0.04 0.06 0.07 0.09
2.44 2.42 2.40 2.34
± ± ± ±
0.05 0.06 0.07 0.05
1.26 1.30 1.21 1.32
± ± ± ±
0.09 0.05 0.04 0.07
1.24 1.21 1.15 1.18
± ± ± ±
0.04 0.06 0.07 0.09
appears to play a crucial role in many cellular processes [46e48]. Intensive studies have confirmed that for most proteins, the thermally denatured states are structurally non-identical to GdmClinduced denatured state [49e51]. Heat-induced denatured states of proteins, in fact, retain lots of residual structure that contains high amount of secondary and tertiary interactions [32,33]. Since residual structure plays an important role in many cellular processes [52], it will be interesting to investigate how cellular osmolytic compounds interact with denatured states containing residual structure and how this interaction is different from that with linear random coils. First of all, we characterized and compared heat-induced and GdmCl-induced denatured states of all the three proteins (barstar, Mb, b-cyt-c) using far-UV CD measurements. We observed that barstar and Mb retain a large amount of secondary structures in their heat-denatured states (Fig. 4A and B) as compared to the GdmCl-induced denatured state. Using the method given by Chen et al. [53], we determined a-helical content retained in the heatinduced denatured state of each protein and compared it with the native protein. We observed that the heat-induced denatured state of Mb retains 23.4% helical structure as compared to 71.8% helical content in the native state. Likewise, the helical content of barstar in the heat denatured state is 11.81% as compared to 41.45% in the native state. The results on barstar and Mb are in good agreements with the earlier reports that heat-induced denatured states of these proteins retain a large amount of secondary and tertiary structures [31,51]. Interestingly, for b-cyt-c, CD spectrum of the thermally denatured state is not significantly different from that of the GdmCl-denatured state (Fig. 4C). The helical content in the denatured b-cyt-c is almost zero (0.6%) as compared to that (34.0%) found in the native state. This finding is in excellent agreement with earlier results showing that GdmCl and heatinduced denatured states are identical in b-cyt-c [49,51]. We conclude that denatured states induced by heat and GdmCl are structurally dissimilar for both barstar and Mb, but is similar in the case of b-cyt-c. 4.2. Osmolyte-induced protein stabilization The thermodynamic basis of the osmolyte-induced protein folding/unfolding (or stabilization/destabilization) has been explained in terms of osmolyte's preferential exclusion from, or preferential binding to the protein surface [54], which is supported by recent observations on the transfer-free energy of the protein groups from the solvent water to the osmolyte aqueous solutions
[55]. Based on these findings, preferential exclusion stabilizes proteins by shifting the denaturation equilibrium, N state 4 D state, towards the left, while preferential binding destabilizes proteins by shifting the denaturation equilibrium toward the right, and therefore making balance of binding and exclusion to be a determining factor for the protein stabilization by an osmolyte. Thus, what effect osmolytes will have on the denaturation equilibrium, N state 4 D state under the native condition could only be understood by measuring DGD in different solvent conditions. The observed DGD values, summarized in Tables 1 and 2, and DDGD versus [osmolyte] plots (Fig. 5A and B), show that there is a clear difference in the extent of protein stabilization (DGD ) by an osmolyte obtained from heat- and GdmCl-induced denaturations of Mb and barstar. If this observed difference in the extent of protein stabilization from the two different modes of denaturation is indeed due to difference in the structural characteristics of the heat- and GdmCl-induced denatured states, then there should not be any difference in DDGD values obtained from GdmCl- and heatinduced denaturation studies in the case of b-cyt-c, for there exists no difference in the structural characteristics of the heat-denatured and GdmCl-denatured states ([49,51]; also see Fig. 4C). Fig. 5C shows that, for each osmolyte, this is indeed true for b-cyt-c, for the measured DDGD values obtained from GdmCl- and heat-induced denaturation measurements are, within experimental errors, identical. All these results strengthen our hypothesis that the variation in the structural characteristics of the denatured state results in the difference in the magnitude of protein stabilization in the presence of osmolytes or protein-osmolyte interaction. It is known from the transfer-free energy measurements that osmolytes interact unfavorably with the peptide backbone exposed upon unfolding (i.e., this interaction forces protein to fold) and favorably with side chains (i.e., this interaction opposes folding). Because GdmCl induces an apparently random coil structure, all the peptide backbones and side chains are fully exposed to the solvent, thereby allowing osmolytes to interact with all the protein groups. Since the number of the peptide group (eNHeCHeCOe) is very much larger than that of side chains interacting favorably with the osmolyte, the unfavorable interaction predominates over the favorable interactions, thereby potentiating the preferential hydration effect in case of random coil polypeptides. Persistence of local structures in the heat-denatured states results in burying of some of the peptide backbones which will be unexposed to the solvent. Consequently, osmolytes will interact with less exposed peptide backbone (leading to low preferential hydration) as compared to the fully exposed polypeptides, and therefore, the
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Fig. 4. Structural characteristics of heat- and GdmCl-induced denatured states of proteins. The far-UV CD spectra of Mb (A), barstar (B) and b-cyt-c (C) were corrected for solvent effect, if any (see text). Solid line, long dashes, and short dashes represent different states of proteins. Inset in the figure shows dependence of mean residual ellipticity at 222 nm on [GdmCl].
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Fig. 6. Experimental and predicted m-values of Mb, barstar and b-cyt-c in the presence of different osmolytes. Gray and black bar represent respectively predicted and experimental m-values of proteins in different osmolytes.
protein groups. These arguments support our observations shown in Fig. 5. This is noteworthy that the osmolyte effect on glutaminyltRNA synthetase from E. coli was shown to depend on the nature of the denatured states of the protein [56]. Fig. 5. DDGD versus [Methylamine] plots of Mb (A), barstar (B), and b-cyt-c (C) at pH 7.0. Curves 1 (TMAO), 2 (sarcosine) and 3 (GB) represent data obtained from GdmClinduced denaturation, and curves 4 (TMAO), 5 (sarcosine) and 6 (GB) represent data obtained from heat-induced denaturation.
magnitude of protein stabilization is less. This therefore suggests that the preferential hydration effects induced by osmolytes on the heat-induced and GdmCl-induced denatured states may not be identical due to possible variations in the exposed and/or buried
4.3. Correlation between m-values and Dai On the molecular level, the degree of unfolding of the heat- and GdmCl-denatured states are directly related to the fractional exposure of amino acid residues of the protein. Therefore, it is important to know how the fractional exposure (Dai) values of the heat- and GdmCl-induced denatured states are related to osmolyteinduced protein stability. The extent of Dai of each residue of a
Table 4 m-values estimated from the linear plots of DDGD versus osmolyte concentration. Mode of denturation
m-value, kcal mol1 M1
Myoglobin
N4D N4D N4D N4D N4D N4D
m-sarcosine
3.77 1.26 4.26 1.24 3.03 1.22
(GdmCl-induced) (Heat-induced) (GdmCl-induced) (Heat-induced) (GdmCl-induced) (Heat-induced)
m-TMAO m-GB
± ± ± ± ± ±
0.13 0.07 0.07 0.04 0.08 0.05
Barstar 2.38 1.40 2.56 1.35 2.32 1.15
± ± ± ± ± ±
0.14 0.06 0.06 0.09 0.16 0.11
b-cyt-c 2.61 2.37 2.80 2.40 2.51 2.32
± ± ± ± ± ±
0.11 0.24 0.15 0.15 0.08 0.14
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protein directly depends on the m-value (i.e., the sensitivity of the DGD of the protein on [osmolyte]). So, estimation of m-value would be an important parameter to correlate the extent of unfolding and the magnitude of stabilization of the protein. The m-value for a protein-co-solvent system can be calculated by the linear analysis of DDGD (co-solvent) versus [co-solvent] plot [19]. It is seen in Table 4 that there is 2e3 fold increase in m-values obtained from the GdmCl-induced denaturation as compared to their heatinduced ones for barstar and Mb. However, there is no significant difference in the observed m-values in the case of b-cyt-c. These observations clearly show that accessible surface area of the denatured state is the determining factor in osmolytes-induced protein stability. Furthermore, on the theoretical basis, Tanford's transfer-free energy model [39] is one method to predict and explain the thermodynamics of protein stability (forcing protein to fold or unfold) in osmolyte solution. The m-value which is a measure of an osmolyte's tendency to force protein to either fold or unfold, can be predicted theoretically using Tanford's transfer-energy model using Bolen's values for the transfer of amino acid residues from water to 1 M osmolyte solution [41,57]. These methods have been successfully employed to predict m-values of a wide range of monomeric, ligand free proteins for a number of osmolytic conditions, and an excellent agreement was found between predicted and experimental data [41,57,58]. To calculate m-value for any protein, first of all we need Dai (fractional change in accessible surface area of the ith type of residue), which can be obtained using Eq. (6). As it is seen in this equation, the evaluation of Da requires ASA of amino acid residues in the denatured state as well as in the native state. The native state solvent accessibility has been obtained from atomic coordinate of these proteins using DSSP server based program [40] but for denatured state solvent accessibility various models has been proposed. These models for the calculation of the denatured state solvent accessibility have been extensively reviewed by Ali et al. [59], and they suggest that the Creamer (LB) and Bernado (maximum) models gave an excellent prediction for m-values. In our case we have used Bernado's denatured state model to calculate theoretical m-values. We have used this model because this model shows very small influence of both the protein size and protein amino acid composition on average ASA of individual residues [42]. Furthermore, Bernado's values for the upper and lower limits can serve to bracket the expected solvent accessibility for the denatured state. It is seen in Fig. 6 that (a) predicted m-values obtained using Bernado's denatured state model, for all three proteins are in agreement with the experimental m-values for two osmolytes sarcosine and TMAO, and (b) in case of glycine-betaine the predicted values were underestimated. At the moment, we ignore the exact reason for this discrepancy, but similar differences in predicted and experimental m-values have been observed in some earlier studies on proteins [60,61].
5. Conclusions We conclude that the extent of protein stabilization by osmolytes, obtained from GdmCl- and heat-induced denaturation measurements, is different due to differences in the structural characteristics of the denatured states induced by these two different denaturants. Caution must, therefore, be taken in comparing an experimental m-value of the protein with the theoretical m-value obtained using Eqs. (5) and (6), for it is possible only when amino acid residues are fully exposed to the solvent on denaturation.
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