International Journal of Biological Macromolecules 72 (2015) 1104–1110
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Transforming growth factor receptor type II (ec-TR II) behaves as a halophile Komal Saini, M. Ashhar I. Khan, Sumit Chakrapani, Shashank Deep ∗ Department of Chemistry, Indian Institute of Technology, Delhi, Hauzkhas, New Delhi, 110016, India
a r t i c l e
i n f o
Article history: Received 10 July 2014 Received in revised form 25 September 2014 Accepted 27 September 2014 Available online 12 October 2014 Keywords: Protein stability Halophile Electrostatic potential
a b s t r a c t The members of transforming growth factor  family (TGF-) are multifunctional proteins but their main role is to control cell proliferation and differentiation. Polypeptides of TGF- family function by binding to two related, functionally distinct transmembrane receptor kinases, first to the type II (TR II) followed by type I receptor (TR I). The paper describes, in details, the stability of wt-ec-TR II under different conditions. The stability of wt-ec-TR II was observed at different pH and salt concentration using fluorescence spectroscopy. Stability of ec-TR II decreases with decrease in pH. Interestingly, the addition of salt increases the stability of the TRII at pH 5.0 as observed for halophiles. Computational analysis using DELPHI suggests that this is probably due to the decrease in repulsion between negatively charged residues at surface on the addition of salt. This is further confirmed by the change in the stability of receptor on mutation of some of the residues (D32A) at surface. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Transforming growth factor beta (TGF-) is a secreted protein that exists in three isoforms called, TGF- I, TGF- II, and TGF- III. It can also act as a negative autocrine growth factor. The members of TGF- family are multifunctional but their main role is to control cell proliferation and differentiation [1,2]. Its down-regulation/upregulation may result in various kinds of diseases, such as cancer and tissue fibrosis. Polypeptides of the TGF- family signal by binding to two related, functionally distinct transmembrane receptor kinases, first to the type II followed by type I receptor [3] and then the signal is sent to the nucleus via SMAD family [4]. TGF- signaling is tightly regulated at different levels along the pathway, and modulation of TGF- receptor activity is a critical step for signaling regulation. Regulation of signaling requires control mechanism and change in pH or salt concentration around the cell may be few among them [5,6,7]. The change in pH and salt concentration may affect the signaling either directly by affecting the interaction or indirectly by affecting the stability of the partners. Loss or gain in stability of receptors may either lead to loss
Abbreviations: TGF-, Transforming growth factor beta; ec-TR II, ectodomain of transforming growth factor receptor II; Tm , Denaturation temperature, Enthalpy of denaturation. ∗ Corresponding author. Tel.: +91 11 26596596; fax: +91 11 26581102. E-mail addresses: [email protected], [email protected] (S. Deep). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.051 0141-8130/© 2014 Elsevier B.V. All rights reserved.
or excess of TGF- function. Thus, it is important to understand the molecular basis of the stability of receptors in TGF- signaling pathway. The purpose of this study is to understand the stability of ectodomain of transforming growth factor receptor II (ec-TR II) in presence of pH and salt. The stability of ec-TR II was observed at different pH and ionic strength using fluorescence spectroscopy. The changes in free energy of stability (G) as a function of pH and salt concentration were also calculated using computational software DELPHI. The calculated and the experimental data were compared to understand the factors governing the stability of the receptor. Interestingly, the receptor shows greater stability in presence of salt. This may be one of the way through which the signaling processes is regulated by the change in environment.
2. Materials and Methods 2.1. Chemicals Receptor type II (ec-TR-II) was expressed and purified as described by Hinck et al. [8,9,10]. Mutants of ec-TR-II were obtained using PCR technique. Luria Broth for expressing ec-TR II was purchased from Himedia (India). Ampicillin, isopropyl-D-thio-galatose (IPTG) and dithiothreitol (DTT) were purchased from Star Microtek. Urea, EDTA, imidazole, sodium acetate, reduced glutathione, oxidized glutathione, Tris-HCl, MES buffer, sodium chloride, acetic acid, sodium dihydrogen phosphate, disodium
K. Saini et al. / International Journal of Biological Macromolecules 72 (2015) 1104–1110
hydrogen phosphate, and glycine were purchased from Sisco Research Laboratories Pvt. Ltd. (India). HCl and NaOH used to maintain pH were also of analytical reagent grade from Sisco Research Laboratories (India). The chemicals were used as such without any further purification. Solutions were prepared in Milli-Q water. The pH was determined on a standard Sartorius (PB-11) pH meter at ambient temperature. Solutions of ec-TR II were prepared via 3× dialysis in the desired buffer and the concentration was determined spectrophotometrically on a Varian Bio 100 spectrophotometer using the extinction coefficient obtained from the software ProtParam tool from ExPASy Proteomics Server (ε280 of 8000 M−1 cm−1 for ec-TR II). The buffer used for the preparation of solutions are: 50 mM phosphate buffer in pH range 6.0–7.0, 50 mM acetate buffer for the range 3.5–5.5, 50 mM glycine buffer for pH 3.0, and 5 mM Tris-HCl buffer for pH 8.0. The pH of the final solution was adjusted by using 1 M HCl or 1 M NaOH.
2.2. CD measurements The conformation of purified ec-TR II was checked by circular dichroism spectra obtained using AVIV Model 420SF circular dichroism spectrometer with temperature controlled peltier. A 1 mm path-length cell was used for the measurement. To obtain the CD spectrum of ec-TR II, protein sample was scanned 5 times, from 200 to 260 nm, in 1.0 nm increment, with a scan rate of 20 nm min−1 , and averaged. The analysis of CD spectra was carried out using CDPro [11] software which includes three different methods for analyzing protein CD spectra implemented in computer programs CDSSTR [12], SELCON3 [13,14], and CONTIN/LL [11,15].
2.3. Stability of wild and mutant proteins using fluorescence The thermal denaturation studies of wild and mutant wt-ecTR II solution (1 M) were carried out by measuring the intrinsic tryptophan fluorescence intensity at max = 333 nm as a function of temperature. For the measurement of tryptophan fluorescence intensity, an excitation wavelength of 295 nm was used with an excitation slit width of 10 nm and an emission slit width of 10 nm. The emission spectra were obtained between 300 and 400 nm. All the measurements were carried out in 10 mm square quartz cells using a Varian Cary Eclipse equipped with peltier temperature controller accessory. A blank measurement recorded prior to protein addition was subtracted from the emission spectra of the protein solutions. A nonlinear least square fit of signal-versus-temperature
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profile was carried out to obtain the thermodynamic parameters of stability using the following equations [16]: K=
y (T ) − yN (T ) , yD (T ) − y (T )
y (T ) =
(1a)
yN (T ) + K × yD (T ) , 1+K
(1b)
G
y (T ) =
yN (T ) + yD (T ) e− RT 1+e
−G RT
.
(1c)
Fluorescence intensity of tryptophan in native and denatured state is temperature dependent [17], and thus Eq. 1c can be expressed as y(T ) =
0 + m T ) + (yo + m T )e (yN N D D
1+e
−G RT
−G RT
.
(1d)
G (T ) [16] is given by
G (T ) = Hm 1 −
T Tm
− CP
(Tm − T ) + T ln
T Tm
,
(1e)
where y (T ) is the fluorescence intensity of the protein at temperature T, G (T ) is the free energy of denaturation at temperature T, Cp is the specific heat capacity of denaturation and Hm is the enthalpy change at Tm , the midpoint of the thermal denaturation, 0 and y0 are the intrinsic tryptophan fluorescence intensity for the yN D native and denatured state at 298 K, T = T − 298, mN and mD are the slope of the linear plot of fluorescence intensity versus temperature for native and denatured state, respectively. Initial values of mN and mD were obtained from their relationship with max [17]. Initial values of other thermodynamic parameters and their confidence intervals (95%) were obtained using method reported by Saini et al. [18]. For the study of chemical denaturation, the fluorescence spectra of protein were collected in presence of increasing concentration of guanidinium hydrochloride (GdmHCl) (emission and excitation wavelength). The stock solution of GdmHCl was prepared in 50 mM phosphate buffer at pH 7.0. The observed fluorescence spectra were then analyzed using Eqs. 1c and 1f. G = G (H2 O) − m [GdmHCl] ,
(1f)
where, m is the measure of the dependence of G on denaturant concentration and G (H2 O) is the estimate of the conformational stability in absence of GdmHCl.
Fig. 1. (a) CD spectra of ec-TR II (16 M, pH 7.0, 25 ◦ C). The obtained spectrum is the average of five scans of the same protein sample. (b) Plot of Gdm Titration of ec-TR II (1 M, pH 7.0).
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Fig. 2. (a) Fluorescence spectra of ec-TR II (pH 7.0) at different temperatures, (b) Near-UV CD spectra of ec-TR II (pH 7.0) at different temperatures, (c) effect of pH on the thermal denaturation profiles of 1 M ec-TRII obtained using fluorescence intensity as probe. The denaturation profiles were analyzed using two state model (Eq. 1d). Table 1 Extracted thermodynamic parameters from the fluorescence thermal profile of 1 M ec-TR II at different pH. CI is 95% confidence interval for various thermodynamic parameters. pH
Denaturation temperature, Tm (K ± CI)
Change on enthalpy of denaturation, Hm (kcal/mol ± CI)
Change on heat capacity of denaturation, Cp (kcal/mol/K ± CI)
4 5 6 7 8
318.0 ± 0.2 324.0 ± 0.1 332.2 ± 0.3 331.1 ± 0.1 330.0 ± 0.2
30.42 ± 5.3 43.50 ± 7.1 43.00 ± 4.8 44.84 ± 6.4 50.92 ± 5.7
0.54 ± 0.36 0.98 ± 0.29 1.05 ± 0.31 1.13 ± 0.42 1.22 ± 0.34
Fig. 3. Plot of change of enthalpy of denaturation versus denaturation temperature at different pH.
2.4. Calculation of electrostatic potential using DELPHI To understand the effect of salt on the stability of protein, the calculations of electrostatic potentials were done by obtaining the solutions of the linear and nonlinear Poisson-Boltzman Eq. using the DELPHI program [19,20]. The dielectric constant inside the molecular surface of the individual proteins as well as complex was assigned a value of 4, while for the solvent outside the molecular surface the assigned value is 80.
3. Results 3.1. Secondary structure of ec-TˇR II The conformation of the purified ec-TR II was checked using CD spectroscopy. Fig. 1a shows the CD spectra of wt-ec-TR II at pH 7.0 and 25 ◦ C. The spectra showed minima at 212 nm. The results using the program SELCON3 [11] shows that there is around 53% beta type structure, 30% random coil while a negligible contribution from alpha type structure. The obtained results are similar to as reported by Deep et al. [21].
3.2. Stability of ec-TˇR II To check the stability of ec-TR II, the fluorescence spectra were recorded in the presence of different concentrations of guanidinium hydrochloride. Fig. 1b represents the normalized intensity at emission maxima (max 333 nm) versus GdmCl concentrations. Fitting of data were carried out using Eqs. 1c and 1f. The obtained value of Cm , m, and Gu are 1.38 ± 0.1 M, 2.72 ± 0.34 kcal/mol.M, and 3.75 ± 0.4 kcal/mol, which is in good agreement with the reported value by De Crescenzo et al. [22]. The stability of the protein was also checked by monitoring the thermal profile of ec-TR II at pH 7. The emission spectra of ecTRII were also collected at different temperatures to look at the change in the environment of tryptophan with temperature Fig. 2a. The fluorescence intensity decreases with increase in temperature and there is a shift of max towards higher wavelength. Increase in max indicates the increase in the polarity of environment around tryptophan on the increase in the temperature [17]. The near-UV CD spectra of the protein can provide information about the packing of aromatic side chains in the protein [23]. The near-UV CD spectrum of ec-TR II shows a distinct
Table 2 Effect of salt concentration (NaCl) on thermodynamic stability of 1 M TR II at different pH. CI is 95% confidence interval for various thermodynamic parameters. Salt concentration [NaCl] (mM)
0 100 200 300 400 500
Denaturation temperature, Tm (K ±CI)
Change on enthalpy of denaturation, Hm (kcal/mol ± CI)
Change on heat capacity of denaturation, Cp (kcal/mol/K ± CI)
pH 4
pH 5
pH 7
pH 4
pH 5
pH 7
pH 4
pH 5
pH 7
318.0 ± 0.2 318.7 ± 0.1 320.9 ± 0.3 321.4 ± 0.2 322.8 ± 0.3 326.2 ± 0.1
324.0 ± 0.1 326.2 ± 0.3 328.2 ± 0.1 329.5 ± 0.2 331.0 ± 0.1 333.5 ± 0.3
331.1 ± 0.1 332.0 ± 0.1 331.7 ± 0.2 330.2 ± 0.1 333.0 ± 0.1 329.3 ± 0.3
30.4 ± 5.3 31.25 ± 4.9 31.77 ± 5.1 34.73 ± 3.5 38.40 ± 7.6 42.64 ± 5.6
43.5 ± 7.1 42.5 ± 6.3 45.0 ± 4.1 48.4 ± 4.7 51.8 ± 5.8 54.9 ± 4.9
41.8 ± 6.4 35.54 ± 7.4 36.90 ± 6.5 40.40 ± 5.9 44.27 ± 6.7 48.40 ± 5.8
0.54 ± 0.36 0.67 ± 0.41 0.73 ± 0.27 0.77 ± 0.39 0.87 ± 0.42 0.90 ± 0.35
0.98 ± 0.29 0.69 ± 0.37 1.02 ± 0.36 0.98 ± 0.41 1.19 ± 0.24 1.37 ± 0.29
1.13 ± 0.42 1.12 ± 0.24 1.50 ± 0.31 1.32 ± 0.28 1.33 ± 0.36 1.57 ± 0.34
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Fig. 4. Thermal fluorescence profiles of wt TRII (1 M) in absence and presence of NaCl. (a) represents the profiles at pH 5 and (b) represents the fluorescence profiles at pH 4.
minimum at 290 nm which is lost on the increase in temperature (Fig. 2b). This suggests a loss in the packing around tryptophan with increase in the temperature. 3.3. Effect of pH on the stability of the ec-TˇR II To understand the physical forces responsible for ec-TR II stability, the thermal profiles of ec-TR II were obtained as a function of pH and salt. The thermal denaturation of ec-TR II is reversible as observed from the thermal profile on reheating. The fluorescence intensity of the same at different pH were recorded as a function of temperature, and shown in Fig. 2c. The extracted thermodynamic parameters obtained from nonlinear fitting are listed
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Fig. 5. Effect of salt concentration ([NaCl]) on thermal fluorescence profile of different mutants i.e., D32A and I50A of ec-TRII (approximately 1 M, pH 5.0).
in Table 1. It was observed that the thermodynamic stability of ecTR II decreases on lowering of pH. However, near physiological pH, there is a negligible change in its stability. The change in heat capacity of denaturation (Cp ) was also determined from the plot of enthalpy change of denaturation versus denaturation temperature at different pH (Fig. 3) and it was calculated to be 1.03 kcal/mol/K. The free energy of stability was calculated to be 2.55 kcal/mol at pH 7 using thermodynamic parameters in Table 1 and Eq. 1e. 3.4. Effect of salt on the stability of the receptor To see the effect of ionic strength on protein stability, thermal fluorescence profile of aqueous solution of ec-TR II was obtained with different salt concentration (NaCl) at pH 4.0 and 5.0 (Fig. 4).
Fig. 6. (a) Stability (Gunfolding ) of wt-ec-TR II as a function of pH. () represents the electrostatic stability while () represents experimental data from fluorescence study. (b) Stability of wt-ec-TR II as a function of salt. () represents the change in free energy of unfolding with ionic strength studied by thermal fluorescence spectroscopy. () represents the electrostatic stability with ionic strength. These values were computed using linear/nonlinear PB method.
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Fig. 7. Change in electrostatic potential of native wt-ec-TR II with pH. Blue portion indicates positive potential while red portion indicates negative potential. (a), (b), and (c) represent front view and (d), (e), and (f) represent back view of native wt-ec-TR II at different pH.
Table 3 Effect of ion types with constant ionic strength on the thermodynamic stability of the wt-ec-TR II (buffer system: acetate buffer, 50 mM, pH 5.0). Salt
Ionic strength (mM)
Transition temperature (K), Tm
Enthalpy of denaturation (kcal/mol), Hm
NaCl KCl MgCl2 NaBr
300 300 300 300
329.5 ± 0.2 328.9 ± 0.1 329.8 ± 0.3 329.1 ± 0.3
48.4 ± 4.7 47.8 ± 5.9 49.9 ± 4.9 47.6 ± 5.2
The extracted thermodynamic parameters are listed in Table 2. It was observed that the effect of ionic strength happens only at low pH. Interestingly, at pH 4 and 5, the stability of the protein, ec-TR II, increases gradually with increase in ionic strength. Tm and Hm increased by nearly 10–12 units with increases in ionic strength from to 0.5 M. To check the origin of salt effect towards the stability, the fluorescence thermal studies were carried out in different salts with constant ionic strength. The results showed that there is no remarkable change in free energy change of unfolding on changing the salt with different cations or anions or changing from monovalent to di-valent without changing the ionic strength and hence Table 4 Effect of salt concentration (NaCl) on thermodynamic stability of D32A and I50A ec-TR II (approximately 1 M, pH 5.0). [NaCl] (M)
D32A 0 100 200 300 400 500 I50A 0 100 200 300 400 500
Denaturation temperature, Tm (K)
Change on enthalpy of denaturation, Hm (kcal/mol)
Change on heat capacity of denaturation, Cp (kcal/mol/K)
327.7 ± 0.1 328.9 ± 0.2 330.2 ± 0.2 331.2 ± 0.1 332.8 ± 0.1 334.2 ± 0.3
40.7 ± 7.8 42.3 ± 6.8 44.6 ± 6.1 46.3 ± 7.2 49.3 ± 5.9 51.5 ± 6.5
0.61 ± 0.24 0.76 ± 0.23 0.99 ± 0.30 0.99 ± 0.28 1.19 ± 0.19 1.24 ± 0.26
320.8 ± 0.2 321.6 ± 0.1 323.4 ± 0.3 325.6 ± 0.1 326.1 ± 0.1 327.5 ± 0.1
35.9 ± 7.7 37.0 ± 5.9 40.2 ± 7.2 40.8 ± 6.4 42.1 ± 6.1 45.2 ± 6.9
0.90 ± 0.35 0.96 ± 0.26 0.96 ± 0.31 1.03 ± 0.19 1.20 ± 0.29 1.21 ± 0.23
Fig. 8. The change in the electrostatic potential and hence stability upon addition of 0.4 M NaCl separated into the contribution from individual residues and projected onto the protein surface. (a) and (b) represents one type of orientation while (c) and (d) shows 180◦ rotation of (a) and (b), respectively.
the increase in stability at high ionic strength can be attributed to nonspecific ion binding (Table 3). 3.5. Stability of the mutants Thermal fluorescence profiles of two mutants of ec-TR II, D32A, and I50A, were obtained at pH 5.0 and in presence of salt at different concentrations (Fig. 5 and Table 4). Similar to the wild type, the stability of the each mutant of ec-TR II increases gradually with increase in ionic strength. The transition temperature and change in enthalpy of denaturation increased nearly 10–12 units with increase in ionic strength from 0 to 0.5 M. 4. Discussion Analysis of thermal profiles of ec- ec-TR II suggests that the receptor possesses moderate stability with transition temperature of 333 K and change in enthalpy of denaturation of 42 kcal/mol at physiological pH. The stability of ec-TR II decreases with decrease in pH and remains unchanged above pH ≈ 6.0 (Fig. 6a).
K. Saini et al. / International Journal of Biological Macromolecules 72 (2015) 1104–1110
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Fig. 9. The change in the electrostatic potential and hence stability of mutated ec-TR II upon addition of 0.4 M NaCl separated into the contribution from individual residues and projected onto the protein surface. (a) represents D32A-ec-TR II while (b) represents I50A-ec-TR II.
The pH dependence of the thermodynamic stability of ec-TR II can be explained on the basis of electrostatic potential in its folded form. The residue-wise contribution to electrostatic potential was calculated based on numerical finite difference solutions of the Poisson-Boltzmann (PB) continuum electrostatic theory [19]. At pH 5 or above, it was found that native state possesses both positive and negative potential which is evenly spread all over the surface (Fig. 7). With decrease in pH, the side chain acidic groups of the amino acid residues get neutralized resulting in highly positive potential on the surface. The dense positive potential around a particular site leads to repulsion resulting in lower stability of protein. This repulsion mainly occurs at three sites comprising of (1) residues 26–31 including L26, K28, F29, D31, (2) residues 45–51 including N46, C47, I49, T50, and (3) residues 115–124 including D117, E118, N120, D121, I123, I124. To gain further insight into physical forces responsible for ecTR II stability, the thermal profiles of ec-TR II were obtained as a function of salt concentration. Interestingly, the stability of ecTR II increases with increase in salt concentration as seen in case of halophilic proteins. Monovalent salts, such as sodium chloride, often affect protein stability by modifying the ionic strength of the solution, which overall can be slightly stabilizing or destabilizing, depending on the specific charge distribution within the protein [24,25]. The electrostatic contribution to the free energy of unfolding was computed using the PB method in the presence of different concentration of salt by calculating the differential electrostatic in its folded and unfolded form. As observed experimentally, the calculations also predict an increase in the stability of ec-TR II on the addition of salt. The comparison between experimental salt profiles with those from the theoretical model is shown in Fig. 6b. These experimental data are found to be in excellent agreement with the theoretical model. To understand the origin of difference in stability on addition of salt, the dissection of the salt effect into per residues contributions is calculated. The surface representations of ec-TR II with potential file corresponding to the residues in folded state at different salt concentration was closely monitored using Visual Molecular Dynamics software (VMD) (shown in Fig. 8). It was found that ecTR II has highly negative charged residues on the surface as in
the case of halophile. The negative charge density (red regions on the surface) resulted in repulsive interactions between the acidic residues which lowers the stability of ec-TRII. As described by M. Mevarech et al. [26], salt reduces the destabilization effect of the electrostatic repulsions in halophilic protein and enhances the hydrophobic effect which allows the solvent to interact with folded form rather than the unfolded form of the protein. In ec-TR II, salt screens the negative electrostatic potential (red region) due to the presence of acidic amino acids residues (D86, E90, D91, E107, and F109) on the surface and thereby stabilizes the protein. This is supported by the observed changed in the stability of ec-TRII on mutation. The stability of D32A is more whereas the stability of I50A is less than the wild type. The surface representations of mutants of ec-TR II in VMD showed that in folded state, the substitution of a negatively charged residue i.e., Asp32 with alanine resulted in decrease in the negative electrostatic potential (reduced red regions on the surface, Fig. 9a). On the other hand, the mutation of a hydrophobic residue i.e., Ile50 to alanine resulted into increase in the negative electrostatic potential at the site (increased red regions on the surface, Fig. 9b) and thereby lower stability of the receptor. On increasing the ionic strength, the stability of both mutants D32A and I50A ec-TR II increases as in case of wild type. Addition of salt tends to reduce these repulsive interactions in the folded form and thereby increasing the stability of the protein.
5. Conclusions The data presented here showed that ec-TR II possesses thermal stability with free energy change of unfolding of 2.55 kcal/mol at pH 7.0. There was a significant decrease in free energy change of unfolding with decrease in pH. This might be due to accumulation of positive charges whose replusive interactions lead to a decrease in the stability of ec-TR II. Salt exhibits nonspecific ion binding with ec-TR II and stabilizes the receptor. Stabilization is due to screening of the repulsive interactions built up within the acidic amino acid residues of a halophilic protein. Thus the salt and pH may regulate the TGF- signaling by affecting the stability of receptor.
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Acknowledgments The authors are thankful to Prof. A. P. Hinck for providing the plasmid of wt-ec-TR II and Dr. C. S. Hinck for providing the protein TGF- III. The authors thank M. S. Nayeem for helpful discussions regarding computational analysis. This work was generously supported by the Department of Science and Technology (DST), Govt. of India (SR/S1/PC-05/2006 dated 16-oct-2006) through grants to S.D. The University Grant Commission (UGC), Govt. of India, generously provided a fellowship to K.S. and A.K. References [1] [2] [3] [4] [5]
D.M. Kingsley, Genes. Dev. 8 (1994) 133–146. H.L. Moses, E.Y. Yang, J.A. Pietenpol, Cell 63 (1990) 245–247. M. de Caestecker, Cytokine Growth Factor Rev. 15 (2004) 1–11. J. Massague, Annu. Rev. Biochem. 67 (1998) 753–791. G. Rabbani, E. Ahmad, N. Zaidi, S. Fatima, R.H. Khan, Cell Biochem. Biophys. 62 (2012) 487–499. [6] G. Rabbani, J. Kaur, E. Ahmad, R.H. Khan, S.K. Jain, Appl. Microbiol. Biotechnol. 98 (2014) 2533–2543. [7] A. Varshney, B. Ahmad, G. Rabbani, V. Kumar, S. Yadav, R.H. Khan, Amino Acids 39 (2010) 899–910. [8] A.P. Hinck, Hinck Laboratory http://hincklab.uthscsa.edu/html/protocols/lab protocols.shtml
[9] F. Chiti, M. Stefani, N. Taddei, G. Ramponi, C.M. Dobson, Nature 424 (2003) 805–808. [10] A.P. Hinck, K.P. Walker, N.R. Martin, S. Deep, C.S. Hinck, D.I. Freedberg, J. Biomol. NMR 18 (2000) 369–370. [11] N. Sreerama, R.W. Woody, Anal. Biochem. 287 (2000) 252–260. [12] W.C. Johnson, Proteins 35 (1999) 307–312. [13] N. Sreerama, R.W. Woody, Anal. Biochem. 209 (1993) 32–44. [14] N. Sreerama, S.Y. Venyaminov, R.W. Woody, Protein Sci. 8 (1999) 370–380. [15] S.W. Provencher, J. Glockner, Biochemistry 20 (1981) 33–37. [16] C.N. Pace, B.A. Shirley, J.A. Thomson, Protein Structure: A Practical Approach, IRL Press at Oxford University Press, Oxford, UK, 1989. [17] K. Saini, S. Deep, Eur. Biophys. J. 39 (2010) 1445–1451. [18] K. Saini, U. Ahluwalia, S. Deep, Thermochim. Acta 506 (2010) 28–33. [19] B. Honig, A. Nicholls, Science 268 (1995) 1144–1149. [20] W. Rocchia, S. Sridharan, A. Nicholls, E. Alexov, A. Chiabrera, B. Honig, J. Comput. Chem. 23 (2002) 128–137. [21] S. Deep, K.P. Walker 3rd, Z. Shu, A.P. Hinck, Biochemistry 42 (2003) 10126–10139. [22] G. De Crescenzo, C.S. Hinck, Z. Shu, J. Zuniga, J. Yang, Y. Tang, J. Baardsnes, V. Mendoza, L. Sun, F. Lopez-Casillas, M. O’Connor-McCourt, A.P. Hinck, Three key residues underlie the differential affinity of the TGFbeta isoforms for the TGFbeta type II receptor, J. Mol. Biol. 355 (2006) 47–62. [23] U. Ahluwalia, S.M. Nayeem, S. Deep, Eur. Biophys. J. 40 (2011) 259–271. [24] Z. Kriz, J. Klusak, Z. Kristofikova, J. Koca, PLoS One 8 (2013) e62914. [25] G. Rabbani, E. Ahmad, N. Zaidi, R.H. Khan, Cell Biochem. Biophys. 61 (2011) 551–560. [26] M. Mevarech, F. Frolow, L.M. Gloss, Biophys. Chem. 86 (2000) 155–164.
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Transforming growth factor receptor type II (ec-TR II) behaves as a halophile Komal Saini, M. Ashhar I. Khan, Sumit Chakrapani, Shashank Deep ∗ Department of Chemistry, Indian Institute of Technology, Delhi, Hauzkhas, New Delhi, 110016, India
a r t i c l e
i n f o
Article history: Received 10 July 2014 Received in revised form 25 September 2014 Accepted 27 September 2014 Available online 12 October 2014 Keywords: Protein stability Halophile Electrostatic potential
a b s t r a c t The members of transforming growth factor  family (TGF-) are multifunctional proteins but their main role is to control cell proliferation and differentiation. Polypeptides of TGF- family function by binding to two related, functionally distinct transmembrane receptor kinases, first to the type II (TR II) followed by type I receptor (TR I). The paper describes, in details, the stability of wt-ec-TR II under different conditions. The stability of wt-ec-TR II was observed at different pH and salt concentration using fluorescence spectroscopy. Stability of ec-TR II decreases with decrease in pH. Interestingly, the addition of salt increases the stability of the TRII at pH 5.0 as observed for halophiles. Computational analysis using DELPHI suggests that this is probably due to the decrease in repulsion between negatively charged residues at surface on the addition of salt. This is further confirmed by the change in the stability of receptor on mutation of some of the residues (D32A) at surface. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Transforming growth factor beta (TGF-) is a secreted protein that exists in three isoforms called, TGF- I, TGF- II, and TGF- III. It can also act as a negative autocrine growth factor. The members of TGF- family are multifunctional but their main role is to control cell proliferation and differentiation [1,2]. Its down-regulation/upregulation may result in various kinds of diseases, such as cancer and tissue fibrosis. Polypeptides of the TGF- family signal by binding to two related, functionally distinct transmembrane receptor kinases, first to the type II followed by type I receptor [3] and then the signal is sent to the nucleus via SMAD family [4]. TGF- signaling is tightly regulated at different levels along the pathway, and modulation of TGF- receptor activity is a critical step for signaling regulation. Regulation of signaling requires control mechanism and change in pH or salt concentration around the cell may be few among them [5,6,7]. The change in pH and salt concentration may affect the signaling either directly by affecting the interaction or indirectly by affecting the stability of the partners. Loss or gain in stability of receptors may either lead to loss
Abbreviations: TGF-, Transforming growth factor beta; ec-TR II, ectodomain of transforming growth factor receptor II; Tm , Denaturation temperature, Enthalpy of denaturation. ∗ Corresponding author. Tel.: +91 11 26596596; fax: +91 11 26581102. E-mail addresses: [email protected], [email protected] (S. Deep). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.051 0141-8130/© 2014 Elsevier B.V. All rights reserved.
or excess of TGF- function. Thus, it is important to understand the molecular basis of the stability of receptors in TGF- signaling pathway. The purpose of this study is to understand the stability of ectodomain of transforming growth factor receptor II (ec-TR II) in presence of pH and salt. The stability of ec-TR II was observed at different pH and ionic strength using fluorescence spectroscopy. The changes in free energy of stability (G) as a function of pH and salt concentration were also calculated using computational software DELPHI. The calculated and the experimental data were compared to understand the factors governing the stability of the receptor. Interestingly, the receptor shows greater stability in presence of salt. This may be one of the way through which the signaling processes is regulated by the change in environment.
2. Materials and Methods 2.1. Chemicals Receptor type II (ec-TR-II) was expressed and purified as described by Hinck et al. [8,9,10]. Mutants of ec-TR-II were obtained using PCR technique. Luria Broth for expressing ec-TR II was purchased from Himedia (India). Ampicillin, isopropyl-D-thio-galatose (IPTG) and dithiothreitol (DTT) were purchased from Star Microtek. Urea, EDTA, imidazole, sodium acetate, reduced glutathione, oxidized glutathione, Tris-HCl, MES buffer, sodium chloride, acetic acid, sodium dihydrogen phosphate, disodium
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hydrogen phosphate, and glycine were purchased from Sisco Research Laboratories Pvt. Ltd. (India). HCl and NaOH used to maintain pH were also of analytical reagent grade from Sisco Research Laboratories (India). The chemicals were used as such without any further purification. Solutions were prepared in Milli-Q water. The pH was determined on a standard Sartorius (PB-11) pH meter at ambient temperature. Solutions of ec-TR II were prepared via 3× dialysis in the desired buffer and the concentration was determined spectrophotometrically on a Varian Bio 100 spectrophotometer using the extinction coefficient obtained from the software ProtParam tool from ExPASy Proteomics Server (ε280 of 8000 M−1 cm−1 for ec-TR II). The buffer used for the preparation of solutions are: 50 mM phosphate buffer in pH range 6.0–7.0, 50 mM acetate buffer for the range 3.5–5.5, 50 mM glycine buffer for pH 3.0, and 5 mM Tris-HCl buffer for pH 8.0. The pH of the final solution was adjusted by using 1 M HCl or 1 M NaOH.
2.2. CD measurements The conformation of purified ec-TR II was checked by circular dichroism spectra obtained using AVIV Model 420SF circular dichroism spectrometer with temperature controlled peltier. A 1 mm path-length cell was used for the measurement. To obtain the CD spectrum of ec-TR II, protein sample was scanned 5 times, from 200 to 260 nm, in 1.0 nm increment, with a scan rate of 20 nm min−1 , and averaged. The analysis of CD spectra was carried out using CDPro [11] software which includes three different methods for analyzing protein CD spectra implemented in computer programs CDSSTR [12], SELCON3 [13,14], and CONTIN/LL [11,15].
2.3. Stability of wild and mutant proteins using fluorescence The thermal denaturation studies of wild and mutant wt-ecTR II solution (1 M) were carried out by measuring the intrinsic tryptophan fluorescence intensity at max = 333 nm as a function of temperature. For the measurement of tryptophan fluorescence intensity, an excitation wavelength of 295 nm was used with an excitation slit width of 10 nm and an emission slit width of 10 nm. The emission spectra were obtained between 300 and 400 nm. All the measurements were carried out in 10 mm square quartz cells using a Varian Cary Eclipse equipped with peltier temperature controller accessory. A blank measurement recorded prior to protein addition was subtracted from the emission spectra of the protein solutions. A nonlinear least square fit of signal-versus-temperature
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profile was carried out to obtain the thermodynamic parameters of stability using the following equations [16]: K=
y (T ) − yN (T ) , yD (T ) − y (T )
y (T ) =
(1a)
yN (T ) + K × yD (T ) , 1+K
(1b)
G
y (T ) =
yN (T ) + yD (T ) e− RT 1+e
−G RT
.
(1c)
Fluorescence intensity of tryptophan in native and denatured state is temperature dependent [17], and thus Eq. 1c can be expressed as y(T ) =
0 + m T ) + (yo + m T )e (yN N D D
1+e
−G RT
−G RT
.
(1d)
G (T ) [16] is given by
G (T ) = Hm 1 −
T Tm
− CP
(Tm − T ) + T ln
T Tm
,
(1e)
where y (T ) is the fluorescence intensity of the protein at temperature T, G (T ) is the free energy of denaturation at temperature T, Cp is the specific heat capacity of denaturation and Hm is the enthalpy change at Tm , the midpoint of the thermal denaturation, 0 and y0 are the intrinsic tryptophan fluorescence intensity for the yN D native and denatured state at 298 K, T = T − 298, mN and mD are the slope of the linear plot of fluorescence intensity versus temperature for native and denatured state, respectively. Initial values of mN and mD were obtained from their relationship with max [17]. Initial values of other thermodynamic parameters and their confidence intervals (95%) were obtained using method reported by Saini et al. [18]. For the study of chemical denaturation, the fluorescence spectra of protein were collected in presence of increasing concentration of guanidinium hydrochloride (GdmHCl) (emission and excitation wavelength). The stock solution of GdmHCl was prepared in 50 mM phosphate buffer at pH 7.0. The observed fluorescence spectra were then analyzed using Eqs. 1c and 1f. G = G (H2 O) − m [GdmHCl] ,
(1f)
where, m is the measure of the dependence of G on denaturant concentration and G (H2 O) is the estimate of the conformational stability in absence of GdmHCl.
Fig. 1. (a) CD spectra of ec-TR II (16 M, pH 7.0, 25 ◦ C). The obtained spectrum is the average of five scans of the same protein sample. (b) Plot of Gdm Titration of ec-TR II (1 M, pH 7.0).
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Fig. 2. (a) Fluorescence spectra of ec-TR II (pH 7.0) at different temperatures, (b) Near-UV CD spectra of ec-TR II (pH 7.0) at different temperatures, (c) effect of pH on the thermal denaturation profiles of 1 M ec-TRII obtained using fluorescence intensity as probe. The denaturation profiles were analyzed using two state model (Eq. 1d). Table 1 Extracted thermodynamic parameters from the fluorescence thermal profile of 1 M ec-TR II at different pH. CI is 95% confidence interval for various thermodynamic parameters. pH
Denaturation temperature, Tm (K ± CI)
Change on enthalpy of denaturation, Hm (kcal/mol ± CI)
Change on heat capacity of denaturation, Cp (kcal/mol/K ± CI)
4 5 6 7 8
318.0 ± 0.2 324.0 ± 0.1 332.2 ± 0.3 331.1 ± 0.1 330.0 ± 0.2
30.42 ± 5.3 43.50 ± 7.1 43.00 ± 4.8 44.84 ± 6.4 50.92 ± 5.7
0.54 ± 0.36 0.98 ± 0.29 1.05 ± 0.31 1.13 ± 0.42 1.22 ± 0.34
Fig. 3. Plot of change of enthalpy of denaturation versus denaturation temperature at different pH.
2.4. Calculation of electrostatic potential using DELPHI To understand the effect of salt on the stability of protein, the calculations of electrostatic potentials were done by obtaining the solutions of the linear and nonlinear Poisson-Boltzman Eq. using the DELPHI program [19,20]. The dielectric constant inside the molecular surface of the individual proteins as well as complex was assigned a value of 4, while for the solvent outside the molecular surface the assigned value is 80.
3. Results 3.1. Secondary structure of ec-TˇR II The conformation of the purified ec-TR II was checked using CD spectroscopy. Fig. 1a shows the CD spectra of wt-ec-TR II at pH 7.0 and 25 ◦ C. The spectra showed minima at 212 nm. The results using the program SELCON3 [11] shows that there is around 53% beta type structure, 30% random coil while a negligible contribution from alpha type structure. The obtained results are similar to as reported by Deep et al. [21].
3.2. Stability of ec-TˇR II To check the stability of ec-TR II, the fluorescence spectra were recorded in the presence of different concentrations of guanidinium hydrochloride. Fig. 1b represents the normalized intensity at emission maxima (max 333 nm) versus GdmCl concentrations. Fitting of data were carried out using Eqs. 1c and 1f. The obtained value of Cm , m, and Gu are 1.38 ± 0.1 M, 2.72 ± 0.34 kcal/mol.M, and 3.75 ± 0.4 kcal/mol, which is in good agreement with the reported value by De Crescenzo et al. [22]. The stability of the protein was also checked by monitoring the thermal profile of ec-TR II at pH 7. The emission spectra of ecTRII were also collected at different temperatures to look at the change in the environment of tryptophan with temperature Fig. 2a. The fluorescence intensity decreases with increase in temperature and there is a shift of max towards higher wavelength. Increase in max indicates the increase in the polarity of environment around tryptophan on the increase in the temperature [17]. The near-UV CD spectra of the protein can provide information about the packing of aromatic side chains in the protein [23]. The near-UV CD spectrum of ec-TR II shows a distinct
Table 2 Effect of salt concentration (NaCl) on thermodynamic stability of 1 M TR II at different pH. CI is 95% confidence interval for various thermodynamic parameters. Salt concentration [NaCl] (mM)
0 100 200 300 400 500
Denaturation temperature, Tm (K ±CI)
Change on enthalpy of denaturation, Hm (kcal/mol ± CI)
Change on heat capacity of denaturation, Cp (kcal/mol/K ± CI)
pH 4
pH 5
pH 7
pH 4
pH 5
pH 7
pH 4
pH 5
pH 7
318.0 ± 0.2 318.7 ± 0.1 320.9 ± 0.3 321.4 ± 0.2 322.8 ± 0.3 326.2 ± 0.1
324.0 ± 0.1 326.2 ± 0.3 328.2 ± 0.1 329.5 ± 0.2 331.0 ± 0.1 333.5 ± 0.3
331.1 ± 0.1 332.0 ± 0.1 331.7 ± 0.2 330.2 ± 0.1 333.0 ± 0.1 329.3 ± 0.3
30.4 ± 5.3 31.25 ± 4.9 31.77 ± 5.1 34.73 ± 3.5 38.40 ± 7.6 42.64 ± 5.6
43.5 ± 7.1 42.5 ± 6.3 45.0 ± 4.1 48.4 ± 4.7 51.8 ± 5.8 54.9 ± 4.9
41.8 ± 6.4 35.54 ± 7.4 36.90 ± 6.5 40.40 ± 5.9 44.27 ± 6.7 48.40 ± 5.8
0.54 ± 0.36 0.67 ± 0.41 0.73 ± 0.27 0.77 ± 0.39 0.87 ± 0.42 0.90 ± 0.35
0.98 ± 0.29 0.69 ± 0.37 1.02 ± 0.36 0.98 ± 0.41 1.19 ± 0.24 1.37 ± 0.29
1.13 ± 0.42 1.12 ± 0.24 1.50 ± 0.31 1.32 ± 0.28 1.33 ± 0.36 1.57 ± 0.34
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Fig. 4. Thermal fluorescence profiles of wt TRII (1 M) in absence and presence of NaCl. (a) represents the profiles at pH 5 and (b) represents the fluorescence profiles at pH 4.
minimum at 290 nm which is lost on the increase in temperature (Fig. 2b). This suggests a loss in the packing around tryptophan with increase in the temperature. 3.3. Effect of pH on the stability of the ec-TˇR II To understand the physical forces responsible for ec-TR II stability, the thermal profiles of ec-TR II were obtained as a function of pH and salt. The thermal denaturation of ec-TR II is reversible as observed from the thermal profile on reheating. The fluorescence intensity of the same at different pH were recorded as a function of temperature, and shown in Fig. 2c. The extracted thermodynamic parameters obtained from nonlinear fitting are listed
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Fig. 5. Effect of salt concentration ([NaCl]) on thermal fluorescence profile of different mutants i.e., D32A and I50A of ec-TRII (approximately 1 M, pH 5.0).
in Table 1. It was observed that the thermodynamic stability of ecTR II decreases on lowering of pH. However, near physiological pH, there is a negligible change in its stability. The change in heat capacity of denaturation (Cp ) was also determined from the plot of enthalpy change of denaturation versus denaturation temperature at different pH (Fig. 3) and it was calculated to be 1.03 kcal/mol/K. The free energy of stability was calculated to be 2.55 kcal/mol at pH 7 using thermodynamic parameters in Table 1 and Eq. 1e. 3.4. Effect of salt on the stability of the receptor To see the effect of ionic strength on protein stability, thermal fluorescence profile of aqueous solution of ec-TR II was obtained with different salt concentration (NaCl) at pH 4.0 and 5.0 (Fig. 4).
Fig. 6. (a) Stability (Gunfolding ) of wt-ec-TR II as a function of pH. () represents the electrostatic stability while () represents experimental data from fluorescence study. (b) Stability of wt-ec-TR II as a function of salt. () represents the change in free energy of unfolding with ionic strength studied by thermal fluorescence spectroscopy. () represents the electrostatic stability with ionic strength. These values were computed using linear/nonlinear PB method.
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Fig. 7. Change in electrostatic potential of native wt-ec-TR II with pH. Blue portion indicates positive potential while red portion indicates negative potential. (a), (b), and (c) represent front view and (d), (e), and (f) represent back view of native wt-ec-TR II at different pH.
Table 3 Effect of ion types with constant ionic strength on the thermodynamic stability of the wt-ec-TR II (buffer system: acetate buffer, 50 mM, pH 5.0). Salt
Ionic strength (mM)
Transition temperature (K), Tm
Enthalpy of denaturation (kcal/mol), Hm
NaCl KCl MgCl2 NaBr
300 300 300 300
329.5 ± 0.2 328.9 ± 0.1 329.8 ± 0.3 329.1 ± 0.3
48.4 ± 4.7 47.8 ± 5.9 49.9 ± 4.9 47.6 ± 5.2
The extracted thermodynamic parameters are listed in Table 2. It was observed that the effect of ionic strength happens only at low pH. Interestingly, at pH 4 and 5, the stability of the protein, ec-TR II, increases gradually with increase in ionic strength. Tm and Hm increased by nearly 10–12 units with increases in ionic strength from to 0.5 M. To check the origin of salt effect towards the stability, the fluorescence thermal studies were carried out in different salts with constant ionic strength. The results showed that there is no remarkable change in free energy change of unfolding on changing the salt with different cations or anions or changing from monovalent to di-valent without changing the ionic strength and hence Table 4 Effect of salt concentration (NaCl) on thermodynamic stability of D32A and I50A ec-TR II (approximately 1 M, pH 5.0). [NaCl] (M)
D32A 0 100 200 300 400 500 I50A 0 100 200 300 400 500
Denaturation temperature, Tm (K)
Change on enthalpy of denaturation, Hm (kcal/mol)
Change on heat capacity of denaturation, Cp (kcal/mol/K)
327.7 ± 0.1 328.9 ± 0.2 330.2 ± 0.2 331.2 ± 0.1 332.8 ± 0.1 334.2 ± 0.3
40.7 ± 7.8 42.3 ± 6.8 44.6 ± 6.1 46.3 ± 7.2 49.3 ± 5.9 51.5 ± 6.5
0.61 ± 0.24 0.76 ± 0.23 0.99 ± 0.30 0.99 ± 0.28 1.19 ± 0.19 1.24 ± 0.26
320.8 ± 0.2 321.6 ± 0.1 323.4 ± 0.3 325.6 ± 0.1 326.1 ± 0.1 327.5 ± 0.1
35.9 ± 7.7 37.0 ± 5.9 40.2 ± 7.2 40.8 ± 6.4 42.1 ± 6.1 45.2 ± 6.9
0.90 ± 0.35 0.96 ± 0.26 0.96 ± 0.31 1.03 ± 0.19 1.20 ± 0.29 1.21 ± 0.23
Fig. 8. The change in the electrostatic potential and hence stability upon addition of 0.4 M NaCl separated into the contribution from individual residues and projected onto the protein surface. (a) and (b) represents one type of orientation while (c) and (d) shows 180◦ rotation of (a) and (b), respectively.
the increase in stability at high ionic strength can be attributed to nonspecific ion binding (Table 3). 3.5. Stability of the mutants Thermal fluorescence profiles of two mutants of ec-TR II, D32A, and I50A, were obtained at pH 5.0 and in presence of salt at different concentrations (Fig. 5 and Table 4). Similar to the wild type, the stability of the each mutant of ec-TR II increases gradually with increase in ionic strength. The transition temperature and change in enthalpy of denaturation increased nearly 10–12 units with increase in ionic strength from 0 to 0.5 M. 4. Discussion Analysis of thermal profiles of ec- ec-TR II suggests that the receptor possesses moderate stability with transition temperature of 333 K and change in enthalpy of denaturation of 42 kcal/mol at physiological pH. The stability of ec-TR II decreases with decrease in pH and remains unchanged above pH ≈ 6.0 (Fig. 6a).
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Fig. 9. The change in the electrostatic potential and hence stability of mutated ec-TR II upon addition of 0.4 M NaCl separated into the contribution from individual residues and projected onto the protein surface. (a) represents D32A-ec-TR II while (b) represents I50A-ec-TR II.
The pH dependence of the thermodynamic stability of ec-TR II can be explained on the basis of electrostatic potential in its folded form. The residue-wise contribution to electrostatic potential was calculated based on numerical finite difference solutions of the Poisson-Boltzmann (PB) continuum electrostatic theory [19]. At pH 5 or above, it was found that native state possesses both positive and negative potential which is evenly spread all over the surface (Fig. 7). With decrease in pH, the side chain acidic groups of the amino acid residues get neutralized resulting in highly positive potential on the surface. The dense positive potential around a particular site leads to repulsion resulting in lower stability of protein. This repulsion mainly occurs at three sites comprising of (1) residues 26–31 including L26, K28, F29, D31, (2) residues 45–51 including N46, C47, I49, T50, and (3) residues 115–124 including D117, E118, N120, D121, I123, I124. To gain further insight into physical forces responsible for ecTR II stability, the thermal profiles of ec-TR II were obtained as a function of salt concentration. Interestingly, the stability of ecTR II increases with increase in salt concentration as seen in case of halophilic proteins. Monovalent salts, such as sodium chloride, often affect protein stability by modifying the ionic strength of the solution, which overall can be slightly stabilizing or destabilizing, depending on the specific charge distribution within the protein [24,25]. The electrostatic contribution to the free energy of unfolding was computed using the PB method in the presence of different concentration of salt by calculating the differential electrostatic in its folded and unfolded form. As observed experimentally, the calculations also predict an increase in the stability of ec-TR II on the addition of salt. The comparison between experimental salt profiles with those from the theoretical model is shown in Fig. 6b. These experimental data are found to be in excellent agreement with the theoretical model. To understand the origin of difference in stability on addition of salt, the dissection of the salt effect into per residues contributions is calculated. The surface representations of ec-TR II with potential file corresponding to the residues in folded state at different salt concentration was closely monitored using Visual Molecular Dynamics software (VMD) (shown in Fig. 8). It was found that ecTR II has highly negative charged residues on the surface as in
the case of halophile. The negative charge density (red regions on the surface) resulted in repulsive interactions between the acidic residues which lowers the stability of ec-TRII. As described by M. Mevarech et al. [26], salt reduces the destabilization effect of the electrostatic repulsions in halophilic protein and enhances the hydrophobic effect which allows the solvent to interact with folded form rather than the unfolded form of the protein. In ec-TR II, salt screens the negative electrostatic potential (red region) due to the presence of acidic amino acids residues (D86, E90, D91, E107, and F109) on the surface and thereby stabilizes the protein. This is supported by the observed changed in the stability of ec-TRII on mutation. The stability of D32A is more whereas the stability of I50A is less than the wild type. The surface representations of mutants of ec-TR II in VMD showed that in folded state, the substitution of a negatively charged residue i.e., Asp32 with alanine resulted in decrease in the negative electrostatic potential (reduced red regions on the surface, Fig. 9a). On the other hand, the mutation of a hydrophobic residue i.e., Ile50 to alanine resulted into increase in the negative electrostatic potential at the site (increased red regions on the surface, Fig. 9b) and thereby lower stability of the receptor. On increasing the ionic strength, the stability of both mutants D32A and I50A ec-TR II increases as in case of wild type. Addition of salt tends to reduce these repulsive interactions in the folded form and thereby increasing the stability of the protein.
5. Conclusions The data presented here showed that ec-TR II possesses thermal stability with free energy change of unfolding of 2.55 kcal/mol at pH 7.0. There was a significant decrease in free energy change of unfolding with decrease in pH. This might be due to accumulation of positive charges whose replusive interactions lead to a decrease in the stability of ec-TR II. Salt exhibits nonspecific ion binding with ec-TR II and stabilizes the receptor. Stabilization is due to screening of the repulsive interactions built up within the acidic amino acid residues of a halophilic protein. Thus the salt and pH may regulate the TGF- signaling by affecting the stability of receptor.
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Acknowledgments The authors are thankful to Prof. A. P. Hinck for providing the plasmid of wt-ec-TR II and Dr. C. S. Hinck for providing the protein TGF- III. The authors thank M. S. Nayeem for helpful discussions regarding computational analysis. This work was generously supported by the Department of Science and Technology (DST), Govt. of India (SR/S1/PC-05/2006 dated 16-oct-2006) through grants to S.D. The University Grant Commission (UGC), Govt. of India, generously provided a fellowship to K.S. and A.K. References [1] [2] [3] [4] [5]
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