In the Laboratory
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Circular Dichroism Method for Heat Capacity Determination of Proteins Cecil L. Jones,*† Chris Bailey, and Kiran Kumar Bheemarti Department of Chemistry, University of South Alabama, Mobile, AL 36688; *
[email protected] The thermodynamic parameters that govern protein unfolding in aqueous solutions are key features to understanding the nature of the solute–solute and solute–solvent interactions. Variables such as the enthalpy, ∆H, entropy, ∆S, and the heat capacity change, ∆Cp, can provide detailed information associated with protein denaturation. Enthalpy changes that occur as a protein unfolds can be used to describe the strength of the intramolecular noncovalent forces (van der Waals, hydrogen bonds, and electrostatic interactions) that stabilize the protein’s native conformation. Changes in entropy may provide information associated with the solvation of a protein and the relative degree of spontaneous change in a given medium (1). The role that solvents play in protein unfolding is crucial, and in many investigations the medium is treated as a ligand bound by the protein. The strength of noncovalent interactions between a protein and solvent and between the different portions of the polypeptide chain have been shown to be both chemically and temperature dependent (2, 3). In the simplest case, the transition between the native, N, and denatured, D, conformations of protein exhibits a two-state mechanism (N D) (4–9). In addition to ∆H and ∆S, the transition midpoint temperature, Tm, for protein unfolding is a key parameter for characterizing this system. Neglecting the effects of aggregation, a description of protein unfolding in a given medium, M, must consider protein–medium interactions
NM
DM
where NM and DM represent the solvated native and denatured conformation of the protein, respectively. The native state is favored only under conditions that give a standard free energy change less than zero (∆G ⬚ < 0). The free energy change depends on both ∆H ⬚ and ∆S ⬚ (1)
∆G ° = ∆H ° − T ∆S °
The equilibrium constant for protein denaturation is given by Kd =
fD fN
where fD and fN are the fractions of the protein in its denatured and native state, respectively. The relationship between ∆G ⬚ and Kd is given by ∆G ° = − RT ln K d
(2)
†
Current address: Department of Natural Sciences & Mathematics, Chemistry Program, Savannah State University, Savannah, GA 31404.
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Calorimetric transition enthalpies for the thermal denaturation of proteins are determined by ∆Hcal° =
Cp dT
(3)
The entropy change can be calculated from the area under the plot of Cp兾T versus T since ∆S ° =
Cp T
dT
(4)
Since ∆H ⬚ and ∆S ⬚ depends on ∆Cp, the free energy change associated with protein unfolding in a given medium ultimately depends on ∆Cp. The heat capacity of an aqueous solution containing a protein undergoing thermal unfolding is a measure of the interaction of aliphatic side chains with water. The ∆Cp for the transfer of these hydrophobic groups from a nonpolar solvent to water at 25 ⬚C is 1.55 J mol᎑1 K᎑1, which is considered to be relatively large (10). The magnitude of ∆Cp for protein unfolding is explained by the formation of extensive hydrogen bonds between water molecules. An increase in the network of hydrogen bonds within water compensates for the absence of favorable electrostatic interactions between the protein and solvent. This exercise will introduce physical and biochemistry students to the thermodynamics of temperature-induced protein denaturation and a spectroscopic determination of ∆Cp. Experimental Procedure The experiment can be completed in two 3-hour laboratory sessions. To conduct thermal unfolding measurements, there must be a spectral difference between the native and denatured conformations of the protein. As an exercise, students should first determine an appropriate concentration of protein sample for spectroscopic work. Two types of concentrations are frequently used for proteins: (i) whole molecule concentration and (ii) per residue molar concentration. The per residue mean molecular weight is given by the whole molecule molecular weight divide by the number of residues (11). For example, bovine pancreatic ribonuclease A (RNase A) weighs about 13,700 Da with 124 amino acid residues giving a per residue mean molecular weight of 110. A solution that is 0.84 mg兾mL has a whole molar concentration of 61.3 µM and a per residue molar concentration of 7.60 mM. This is a typical concentration for thermal unfolding studies. A Jasco 810 circular dichroism (CD) spectropolarimeter was used to measure the thermal denaturation of RNase A. It is important to note that a relatively inexpensive UV–vis spectrophotometer (Cary 100) equipped with a temperature
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In the Laboratory Table 1. Thermodynamic Parameters Associated with the Thermal Unfolding of RNase A with Various Concentrations of GuHCl
Figure 1. CD spectra of RNase A illustrates three regions where thermal unfolding of the protein may be observed. The smaller plot shows the spectral difference in the near UV region.
Figure 2. A red shift in the transition midpoint temperature, Tm, for RNase A unfolding occurs with increasing concentration of GuHCl.
GuHCl/M
Tm/°C
∆H/(kJ mol᎑1)
∆S/(J mol᎑1 K᎑1)
0.0
60.1
382
1151
0.5
56.2
368
1117
1.0
50.0
344
1063
1.5
45.5
325
1021
2.0
39.1
297
0954
ramping device can perform the same measurements. All chemicals were obtained from Sigma Chemical Company. A solution of 61 µM RNase A in 50 mM potassium acetate buffer at pH 5.5, conditions suitable for investigating protein–ligand interactions (12), was prepared. An appropriate wavelength for monitoring the thermal unfolding of the protein was determined. A 0.1-cm cell was used to measure a CD spectrum for 61 µM RNase A at 25 ⬚C from 190 to 300 nm. This spectrum was compared with another one measured at 80 ⬚C as RNase A is completely denatured at 80 ⬚C (Figure 1). The wavelength of 280 nm was selected to monitor the transition of RNase A from the native to its denatured conformation. A 1.0-cm cell worked well at this wavelength. Solutions of 61 µM RNase A in 0.0, 0.5, 1.0, 1.5, and 2.0 M guanidine hydrochloride (GuHCl) were prepared. The thermal unfolding of each RNase A兾salt sample over the temperature range of 25–80 ⬚C were measured. An overlay of these measurements produced a red shift in Tm as the concentration of GuHCl increased (Figure 2). Varying the condition of the medium can cause either a blue or red shift in Tm depending on the nature of interfering solute. For example, neutral salts like NaCl are generally known to stabilize the native conformation of proteins requiring higher temperature for denaturation (Figure 3). The addition of small quantities of NaCl consequently caused a shift in Tm to higher temperatures (blue shift). Computer software for analyzing the thermal unfolding curves is usually provided with instruments capable of monitoring the thermal denaturation of proteins. The data were fit to obtain the Tm and ∆H for each sample (Table 1). Graphs of Tm and ∆H versus the concentration of GuHCl were obtained (Figures 4 and 5). The value of ∆Cp was calculated from the slope of a plot of ∆H versus Tm (Figure 6), which is obtained from eq 3 through differentiating and rearrangement. Hazards
Figure 3. The transition midpoint temperature, Tm, for the thermal denaturation of RNase A is blue shifted with increasing concentration of NaCl.
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Guanidine hydrochloride (GuHCl) is toxic. Protective gloves and goggles should be used throughout the experimental procedure. Enzymes and proteins may cause allergic reactions in some sensitive individuals. Allergy-prone and asthmatic individuals should exercise caution when working with RNase A. A dust mask or respirator in addition to protective gloves and goggles will minimize RNase A contact with sensitive students.
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In the Laboratory
Results and Discussion 60
Tm / °C
55
50
45
40
35 0.0
0.5
1.0
1.5
2.0
GuHCl Concentration / (mol/L) Figure 4. Effects of increasing GuHCl concentration on the transition midpoint temperature, Tm, of RNase A unfolding.
⌬H / (kJ/mol)
380
360
ln K d = −
340
320
300
0.0
0.5
1.0
1.5
2.0
GuHCl Concentration / (mol/L) Figure 5. Effects of increasing GuHCl concentration on the enthalpy change of RNase A unfolding.
380
⌬H / (kJ/mol)
In the absence of calorimetric data, the van Hoff enthalpy change (∆H ⬚) can be determined from the thermal unfolding of a protein that exhibits a reversible two-state mechanism. Spectroscopic instruments designed to do thermal melting frequently come with software that calculate thermodynamic parameters associated with protein unfolding. We wrote a computer program called “Thermal Fit” that allowed us to fit the data in the software package, MatLab. The program was designed to produce a calculated curve based on the two-state model that describes the equilibrium between the native and denatured states of the protein (3). An optimum fit was obtained by minimizing the difference (sum of the squared residuals) between the experimental and computer calculated curve. Between the range of 25 and 45 ⬚C, RNase A is in its native state and can be fitted to a linear dependence on temperature. This was also done for the denatured state of the protein measured from 70 to 80 ⬚C. Thermal Fit provides Tm in addition to the fraction of the protein in the native and unfolded conformation over the measured temperature range. The program then generates a plot for the natural logarithm of Kd versus 1兾T in the linear region where the change in signal versus T is the greatest. This corresponds to the temperature region where Tm is calculated. Equations 1 and 2 can be combined to give
360
340
∆H ° ∆S ° + RT R
(5)
The values for ∆H ⬚ and ∆S ⬚ are provided by the slope and y intercept, respectively. The value of ∆Cp for the thermal denaturation of RNase A was determined from (Figure 6) to be 4.0 kJ mol᎑1 K᎑1 with 2.0% relative error. Reported ∆Cp values for RNase A unfolding range from 4.2 to 9.6 kJ mol᎑1 K᎑1. Makhatadze and co-workers reported a value of 5.0 kJ mol᎑1 K᎑1 (13). The effect of GuHCl on proteins has been investigated extensively and is characterized as a strong protein denaturant. Destabilizing effects of GuHCl on the native conformation of RNase A can be observed from Figure 4 where d(Tm)兾d([GuHCl])is substantial giving a value of ᎑10.5 ± 0.5 ⬚C兾M. This exercise should invite questions regarding the mechanism by which GuHCl destabilizes proteins. Explanations can range from water–water interactions to binding with hydrophobic side chains (14). While differential scanning calorimeter represents a direct method for measuring the increase in ∆Cp for protein unfolding (15), a spectroscopic method offers the opportunity for students to observe the spectral effects of both temperature and ligand-induced protein denaturation.
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Supplemental Material
300 35
40
45
50
55
60
Tm / °C Figure 6. A plot of the change in enthalpy versus the transition midpoint temperature, Tm, resulting from the addition of GuHCl to RNase A.
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Notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Creighton, T. Proteins: Structures and Molecular Properties; W. H. Freeman: New York, 1988; pp 132–152.
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In the Laboratory 2. Loladze, V.; Ermolenko, D.; Makhatadze, G. Protein Science 2001, 10, 1343–1352. 3. Jones, C.; Muccio, D.; Fish, F. Anal. Biochem. 2002, 302, 184– 190. 4. Kumar, S.; Tsai, C.; Nussinov, R. Biochem. 2002, 41, 5359– 5374. 5. Brandts, J.; Lin, L. Biochem. 1990, 29, 6927–6940. 6. Ramsay, G.; Eftink, M. Biophys. J. 1994, 31, 516–523. 7. Schwarz, F. Biochem. 1988, 27, 8429–8436. 8. Nussinov, R.; Tsai, C.; Sandeep, K. Biochem. 2002, 41, 5359– 5374. 9. Pace, C. Crit. Rev. Biochem. 1975, 3, 1–43.
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10. Ooi, T.; Oobataka, M. J. Biochem. 1988, 103, 114–120. 11. Protein–Ligand Interactions: Structure and Spectroscopy; Harding, S., Chrowdhry, B., Eds.; Oxfod University Press: Oxford, 2001; pp 123–167. 12. Anderson, D.; Hammes, G.; Walz, F. Biochem. 1968, 7, 1637– 1645. 13. Makhatadze, G.; Privalov, P. Adv. Prot. Chem. 1995, 47, 307. 14. Nishimura, C.; Uversky, V.; Fink, A. Biochem. 2001, 40, 2113– 2128 15. Pace, N.; Grimsley, G.; Thomas, S.; Makhatadze, G. Protein Science 1999, 8, 1500–1504.
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