Osmolyte Counteracts Urea-Induced Denaturation of Î±-Chymotrypsin

J. Phys. Chem. B 2009, 113, 5327–5338

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Osmolyte Counteracts Urea-Induced Denaturation of r-Chymotrypsin Pannur Venkatesu,† Ming-Jer Lee,* and Ho-mu Lin Department of Chemical Engineering, National Taiwan UniVersity of Science and Technology, 43 Keelung Road, Section 4, Taipei 106-07, Taiwan ReceiVed: December 22, 2008; ReVised Manuscript ReceiVed: February 4, 2009

The stability of proteins is reduced by urea, which is methylamine and nonprotecting osmolyte; eventually urea destabilizes the activity and function and alters the structure of proteins, whereas the stability of proteins is raised by the osmolytes, which are not interfering with the functional activity of proteins. The deleterious effect of urea on proteins has been counteracted by methylamines (osmolytes), such as trimethylamine N-oxide (TMAO), betaine, and sarcosine. To distinctly enunciate the comparison of the counteracting effects between these methylamines on urea-induced denaturation of R-chymotrypsin (CT), we measured the hydrodynamic diameter (dH) and the thermodynamic properties (Tm, ∆H, ∆GU, and ∆Cp) with dynamic light scattering (DLS) and differential scanning calorimeter (DSC), respectively. The present investigation compares the compatibility and counteracting hypothesis by determining the effects of methylamines and urea, as individual components and in combination at a concentration ratio of 1:2 (methylamine:urea) as well as various urea concentrations (0.5-5 M) in the presence of 1 M methylamine. The experimental results revealed that the naturally occurring osmolytes TMAO, betaine, and sarcosine strongly counteracted the urea actions on R-chymotrypsin. The results also indicated that TMAO counteracting the urea effects on CT was much stronger than betaine or sarcosine. Introduction Naturally occurring osmolytes are small organic molecules, which comprise the bulk of the osmotically active solutes, accumulated by many organisms and cells in response to osmotic stress.1,2 These osmoregulatory compounds have also been shown to help cells coping with other environmental stresses such as high temperature, pressure, or desiccation and to affect the stabilities of proteins.3,4 Osmolytes belong to diverse chemical families including polyols, amino acids, and amino acid derivatives and methylamines.2,5,6 These osmolytes, referred to as compatible or protective osmolytes, tend to stabilize the protein structure, without perturbing macromolecular structure and functions through unfavorable interactions with the surface of the protein.7,8 However, urea, the principal noncompatible osmolyte and also methylamine, is a denaturant. The unfolding of proteins by urea, which is a polar molecule, has long been considered to arise because of the favorable interaction of this denaturant with the normally buried interior segments of a protein.9,10 On the other hand, the third group of nitrogenous osmolytes, the methylamines, acts by stabilizing the folded state and counteracting the perturbations of protein structure caused by urea. Counteracting or compensatory methylamines, such as trimethylamine N-oxide (TMAO), betaine (glycine betaine), and sarcosine, appear to offset the deleterious effects of urea catalytic activity and permit normal cellular function by stabilizing proteins.2,9,11-22 The counteracting effect can be viewed as a situation in which unfolded protein structure is counterbalanced by a tendency of the methylamine to minimize the protein surface area in contact with water.21,23 Moreover, Yancey7 * To whom correspondence should be addressed. Tel.: +886-2-27376626. Fax: +886-2-2737-6644. E-mail: [email protected] (M.-J.L.); [email protected] (P.V.). † Current address: Department of Chemistry, University of Delhi, Delhi 110 007, India.

reported that TMAO was able to increase the protein melting temperature and also counteract the temperature-perturbing actions on proteins. Knowledge of the detailed functioning of enzymes is crucial for understanding their metabolic role and their use as industrial biocatalysts. The serine proteinases are widely distributed in nature, where they perform a variety of different functions. Many proteinases occur as domains in large multifunctional proteins, but others are independent smaller polypeptide chains.24 Bacterial serine proteinases share the chymotrypsin-like bilobal β-barrel structure, but are more distantly related due to their shorter sequences and structural differences in surface loops.25-31 Proteolytic enzyme R-chymotrypsin (CT), which has a potential for use in industrial applications, is composed into two juxtaposed β-barrel domains, with catalytic residues bridging and the disulfide bridges that join the three polypeptide chains. The crystal structure of CT is shown in Figure 1. The polypeptide chain of CT comprises 245 amino acids and catalytic triad is formed by His 57, Asp 102 and Ser 195, which is reactive group and part of the second domain.24,25 Virtually, the crystalline structure of CT has total five disulfide bonds25(protein data bank, ID code 2CHA). Out of five disulfides, two (Cys1-Cys122 and Cys42-Cys58) are close to the enzymatic active site, which catalytic center and one (Cys191-Cys220) is close to the well-known surface binding site, which located in vicinity of catalytic triad for the formation of binding. The rest of the two disulfide bonds (Cys136-Cys201 and Cys168-Cys182) are away from both enzymatic sites (protein data bank) (Figure 1). CT is one of the valuable biological substances for understanding the mechanism of protein folding or unfolding with the addition of cosolvents.32 Nevertheless, understanding of the structure-function relationship is still one of challenging tasks in biochemical, biotechnology and protein science. The enzyme surface is initially responsible for the interaction with the environment of the

10.1021/jp8113013 CCC: $40.75  2009 American Chemical Society Published on Web 03/23/2009

5328 J. Phys. Chem. B, Vol. 113, No. 15, 2009

Figure 1. Schematic diagram of the crystal structure of CT, which is downloaded protein data bank (ID code 2CHA) and processed with PyMOL viewer software. The catalytic triad in the hydrophobic pocket is located at the middle part of the right-hand side of the structure. The stick models are used to indicate five disulfide bonds of the enzyme.

solvent particles. The possibility of enzymatic reactions in solvent media is interesting from both theoretical and practical points of view to estimate the protein folding problem.33 Osmolyte effects have initially focused by Yancey et al.2 on protein folding and enzyme functions. Many growing different strategies to increase CT stability have been assayed by several researchers.34-42 Moreover, a great deal of work has well explored by various research groups31,43-47 to understand the changes in structure and unfolding of CT by the addition of denaturants, urea and Guanidinium salts. However, there has been no systematic documentation of the influence of the osmolytes on CT to understand the thermal stability as well as the thermal denaturation of urea actions on CT. Obviously, a good deal of effort has been directed toward understanding the counteraction of TMAO against the deleterious effects of urea in a variety of protein systems 14-18 effectively at the molar ratio 1:2 of TMAO:urea. It was concluded that TMAO strongly counteracted the urea actions on proteins at this condition. However, no conclusive results have been explored systematically that the counteracting effects of TMAO (1 M) or the rest of two methylamines (1 M), in the presence of higher urea concentrations on perturbing urea actions on proteins. Moreover, there is a lag of conclusions in the earlier studies that TMAO opposes urea actions on proteins only at 1:2 of TMAO:urea. Apparently, 1:2 ratio may not be a physiological ratio.7,48 For instance, in our earlier studies, we found that TMAO (1 M) strongly counteracted the urea deleterious actions on cyclic dipeptides (CDs) in the presence of higher urea concentrations while TMAO counteracted only at 1 M TMAO:2 M GdnHCl and failed to counteract GdnHCl actions on CDs at higher concentrations (3-6 M) of GdnHCl. However, a comparison of the counteracting effects of the methylamines (TMAO, betaine, and sarcosine) against the perturbations caused by urea on the proteins has received less attention.49 Moreover, there has been no systematic documentation data of compatibility as well as counteracting effects of methylamines on the temperature perturbing and urea deleterious actions on proteins by the great versatility of technique of dynamic light scattering (DLS) as well as differential scanning calorimeter (DSC) experiments. With these considerations in mind as mentioned above and in the view of their importance, it is of interest to compare the counteracting effects of

Venkatesu et al. methylamines, such as TMAO, betaine, and sarcosine against the urea as well as temperature perturbing actions on proteins. In order to establish a comparison of compatibility and counteracting effects between methylamines on the urea-induced denaturation of CT and the temperature deleterious action of CT, we measured the hydrodynamic diameter (dH) and the calorimetric data (Tm, ∆H, and ∆Cp) by using DLS and DSC, respectively, at various concentrations of TMAO, betaine, sarcosine, urea, individually and their mixtures containing the molar ratio of 1:2 of methylamine:urea and varying the urea concentrations (from 1 to 5 M) in the presence of 1 M methylamine. To check the methylamine offset effects against the temperature perturbation effect on the enzyme CT, we used 1 M methylamine and 1 M urea as a function of temperature through the DLS measurements. Additionally, we have estimated the Gibbs free energy of unfolding changes (∆GU) at 25 °C, which is a better indication of global protein stability, from thermal melting analysis, enthalpy (∆H) and heat capacity (∆Cp) data. Materials and Methods R-Chymotrypsin (CT) from bovine pancreas type II, essentially salt free (molecular weight, 25 kDA) was obtained from Sigma-Aldrich. The osmolytes TMAO and betaine were supplied by Sigma Chemical Co. Sarcosine was purchased from Fluka Biochemical Co., urea from Acros Organics, and tris(hydroxymethyl)aminomethane from Aldrich Chemical Co. All materials, with high purity, were used without further purification. Buffer solution was prepared using distilled deionized water at 18.3 MΩ. All mixture samples were prepared gravimetrically. Enzyme Stability. Enzyme stability was analyzed by incubating 2 mL screw-capped vials in 0.05 M Tris-HCl buffer pH 8.20 solutions in the presence or the absence of methylamine, urea or methylamine + urea mixture of various concentrations at 25 °C for 4 h to attain complete equilibrium. All samples were prepared at 15 mg/ml enzyme concentration for DLS as well as DSC experiments. After completely dissolving the enzyme in the solution, the mixture was filtered with a 0.45 µm disposal filter (Millipore, Millex-GS) through syringe before performing the measurements. Dynamic Light Scattering Experiments. Dynamic light scattering (DLS) measurements have been made with Zetasizer Nano ZS90 (Malvern Instruments Ltd.) for determining the diameter of the sample. This instrument employs a 4 mW He-Ne laser with a fixed wavelength (λ ) 633 nm) and is equipped with a thermostatted sample chamber for maintaining the desired temperatures within a temperature range of 2-90 °C. DLS instrument has one detector only detects the scattered light at a fixed scattering angle, usually at 90 °C. A bubblefree sample of around 2 mL was introduced in a quartz cuvette (QC) sample cell through a syringe. The cell was then sealed airtight with a Teflon-coated screw cap and secured in a sample chamber of DLS. The diameter measured with DLS is denoted as the hydrodynamic diameter (dH), which refers to how a particle diffuses within a fluid. The scattering intensity data were processed using the instrumental software to obtain the hydrodynamic diameter and the size distribution of the sample. The value of dH was calculated by using the Stokes-Einstein equation:

dH )

kT 3πηD

(1)

where k is the Boltzmann constant, T is absolute temperature, η is the viscosity of the sample, and D is the translational

Urea-Induced Denaturation of R-Chymotrypsin

Figure 2. DLS spectra of intensity distribution graph as a typical size distribution in nanometers of CT in buffer solution at various temperatures: (O) 15, (∆) 25, (0) 35, (3) 45, (b) 55, (2) 65, (9) 75, and (1) 85 °C.

diffusion coefficient, which was obtained from the intensity autocorrelation function of the time-dependent fluctuation in intensity. Differential Scanning Calorimeter. A differential scanning calorimeter (DSC; N 520-0092, Perkin-Elmer Instruments) was used to measure the melting point of CT in the different solvent media as described above. A certain amount of bubble-free solution was placed into a 0.1 mL of DSC sample cell, while 0.1 mL of the reference cell was filled with an appropriate blank sample of the same solvent media without CT, then capped, and sealed using a press. The sample mass is in the range of 10-30 mg. The cells were allowed to stabilize at 20 °C inside the calorimeter before heating to 95 °C with a scanning rate of 1.0 °C/min. Calorimetric heat flow (H) and the melting temperature (Tm) of the sample were determined from Pyris Software, which was included in the DSC. The uncertainties of temperature and heat flow readings are (0.02 °C and (0.1%, respectively. To obtain accurate results, the instrument has been calibrated with pure water and sapphire. Results and Discussion To check the temperature perturbation effect on the enzyme CT, we have performed DLS measurements by using 1 M methylamine and 1 M urea as a function of temperature. Figure 2, which is an intensity distribution graph, depicts the relative intensity of the scattered light from the DLS measurements of CT in the buffer solution at various temperatures as a typical size distribution in nanometers. The measured intensity autocorrelation function g(τ) using DLS for CT in the buffer solution is displayed in Figure 3 as a function of time. For the sake of clarity we do not present the rest of the intensity distribution graphs and g(τ) of CT in 1 M methylamine or 1 M urea as a function of temperature. However, Table 1 and Figure 4 depict the particle size (dH) of CT in the presence or the absence of 1 M methylamine or 1 M urea of 0.05 M Tris-HCl buffer, pH 8.20, solution as a function of temperature, which was obtained from an intensity distribution graph of CT in buffer (from Figure 2), 1 M methylamine, or 1 M urea (we did not shown here). As noted from Figure 2, the signal is polydisperse and there are two peaks at the native state of CT at lower temperatures (from 15 to 45 °C). Note that in the intensity distribution graph, our samples’ polydispersity indexes (PDIs)50 do not exceed 0.5,

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Figure 3. Measured intensity autocorrelation function g(τ) using DLS for CT (native and denatured) in buffer solution at various temperatures: (O) 15, (∆) 25, (0) 35, (3) 45, (b) 55, (2) 65, (9) 75, and (1) 85 °C.

actually between 0.115 and 0.281. Our experimental results in Figure 2 and Table 1 explicitly show that dH values of CT in the buffer solution are from 11.6 to 14.4 nm, which were obtained from a major population (i.e., native state) at temperatures from 15 to 42 °C. As seen from the intensity distribution graph, lower percentages of intensity (from 5.50 to 8.05%) were observed for the second peaks (at temperatures from 15 to 45 °C), which are obviously smaller than the those of major peaks at 15-42 °C, and, thereby, particle sizes of the second populations are negligibly small.51,52 Moreover, the second population (the second peak) shows that the CT molecules are highly aggregated (dH is 150 nm or greater) in the solution. It is also noted that in the intensity distribution graph the area of the peak for high aggregation will appear at least 106 times larger than that of the first peak for smaller particles. The intensity of scattering of a particle is proportional to the sixth power of its diameter, according to the Rayleigh’s approximation. Thus, the larger particles in the second population are negligibly small.53,54 It is quite clear from Figure 2 the native state peaks sharply decrease as well as the growth of the large peak (denature state) increases with increasing temperature. Eventually, we observed single peaks at higher temperatures (55-85 °C), and absolutely the native populations had vanished. These results indicate that dH values increase with increasing temperature, slowly below 45 °C but much more rapidly above 45 °C. The values of ,dH rapidly increase with increasing temperature especially at 55-85 °C, and it reaches 342 nm at 65 °C for CT in the buffer solution, indicating the growth of the denaturing peak. Such high values of dH could be argued as being due to the aggregation of the protein upon unfolding.55-57 Figure 3 shows that dH values are obviously small at lower temperatures and are appreciably larger at higher temperatures as a function of time. It was observed that the values of g(τ) significantly decrease as expected with an increase of crystallization time up to 3000-3500 µs and then there is no significant change in g(τ). Counteracting Effects of Methylamines and Urea on Disruption of CT Structure by Temperature. DLS measurements were performed at various temperatures for CT in the buffer, 1 M methylamine, and 1 M urea to obtain the absolute value of hydrodynamic diameter. The results in Figure 4 distinctly enunciate that the native protein in the buffer solution has a dH ) 12.1 nm at 25 °C. The diameter is not appreciably changed; the value of dH remains constant up to 42 °C. Then a


Venkatesu et al.

TABLE 1: DLS Data of Hydrodynamic Diameter (dH) for r-Chymotrypsin in Various Solutions at Different Temperatures solvent buffer

1 M TMAO

1 M betaine

a

T (°C)

dHa (nm)

15 25 35 40 42 45 46 47 50 55 65 75 85 15 25 35 40 45 46 47 48 49 50 51 55 65 75 85 15 25 30 32 35 40 42 44 45 46 47

11.6 ( 0.15 12.1 ( 0.09 12.5 ( 0.10 13.6 ( 0.25 14.4 ( 0.47 40.2 ( 0.14 100 ( 10.5 177 ( 12.20 214 ( 8.90 256 ( 12.40 342 ( 13.44 526 ( 28.69 695 ( 78.23 14.1 ( 0.05 14.5 ( 0.30 14.7 ( 0 0.07 14.3 ( 0.34 14.5 ( 0.15 14.8 ( 0.48 18.0 ( 0.61 29.6 ( 0.72 47.8 ( 0.25 82.0 ( 2.50 134 ( 5.46 203 ( 14.10 265 ( 19.00 479 ( 55.56 700 ( 85.23 15.2 ( 0.21 15.4 ( 0.15 15.8 ( 0.53 15.6 ( 0.57 15.9 ( 0.56 16.6 ( 0.20 18.6 ( 0.70 19.2 ( 0.82 25.7 ( 0.18 49.9 ( 0.84 61.8 ( 2.08

solvent

1 M sarcosine

1 M urea

T (°C)

dHa (nm)

48 49 50 55 65 75 85 15 25 35 40 42 43 44 45 46 47 48 49 50 55 65 75 85 15 25 30 32 35 36 38 39 40 42 45 55 65 75 85

137 ( 4.66 174 ( 4.80 200 ( 7.40 243 ( 5.08 312 ( 15.00 522 ( 45.96 815 ( 92.02 16.1 ( 0.24 16.4 ( 0.22 16.9 ( 0.26 17.4 ( 0.14 18.3 ( 0.26 19.1 ( 0.31 30.4 ( 0.32 35.0 ( 1.01 46.7 ( 0.73 108 ( 2.02 134 ( 1.62 170 ( 3.47 186 ( 3.76 233 ( 7.52 303 ( 14.2 575 ( 52.36 836 ( 89.31 31.9 ( 0.52 32.3 ( 0.65 33.0 ( 0.45 35.6 ( 0.42 38.1 ( 0.75 43.9 ( 0.95 65.6 ( 0.75 96.3 ( 9.50 134 ( 4.29 177 ( 6.21 215 ( 17.00 334 ( 8.60 410 ( 9.20 650 ( 99.23 910 ( 100.20

Each sample run we performed five times. This uncertainty indicates the standard deviation of the measurements.

Figure 4. Hydrodynamic diameter (dH) obtained from the intensity distribution graph for CT in buffer (control) (O), 1 M TMAO (∆), 1 M betaine (0), 1 M sarcosine (b), and 1 M urea (2) as a function of temperature. For the sake of clarity we did not provide the particle sizes at 75 and 85 °C, since we observed larger values.

significant increase of dH was observed beyond 42 °C. This indicates that the CT is in a folded form up to 42 °C and the CT starts to denature with increasing temperature. The diameter

rapidly increases with increasing temperatures (for example, 342 nm at 65 °C), revealing that the protein was swelling and completely unfolded beyond 42 °C. When the system was cooled from 85 to 25 °C, then we observed that dH did not recover its original value, as evidence of our DLS measurements. Interestingly, it can be seen that, from this graph, in the presence of 1 M TMAO the change in the native structure of CT against temperature is quite sharp beyond 47.5 °C and the enzyme started thermal denaturation beyond 47.5 °C, indicating that TMAO promoted CT thermal stability up to 47.5 °C, which is significantly higher than that in the buffer (control) solution (42 °C). It is clear that TMAO keeps going on the folded form up to higher temperature (47.5 °C), and it protects the native structure of CT against the perturbation of temperature. Furthermore, Figure 4 elucidates that betaine and sarcosine are also able to offset effects against the thermal denaturation on CT. Betaine and sarcosine increase the stability temperatures approximately up to about 44 and 43 °C, respectively, which are obviously higher than that of the control value (42 °C). Taken together, these results indicate that methylamines suppress the aggregation of a thermal denatured protein. The results reported here explicitly indicate that methylamines have comparable effects on counteracting the temperature-perturbing actions on CT. The counteracting effects of TMAO against the temperature-perturb-



Figure 5. Counteracting effects of methylamine against urea-induced denaturation of CT. The hydrodynamic diameter (dH) at 25 °C for CT in methylamines (O) [(a) TMAO, (b) betaine, and (c) sarcosine] and urea (∆) as well as the combination of various ratios of methylamine:urea (b). The point 12.1 ( 0.09 nm is control, which is the buffer solution. For the sake of clarity presentation we did not show the value of dH ) 235 ( 12.65 nm for CT in 6 M urea solution. The dashed lines show the region of methylamine + urea.

ing actions on CT are similar to those of betaine or sarcosine. However, TMAO (thermal stability temperature, 47.5 °C) that actually attenuated the effects caused by temperature on the enzyme seems more effective than betaine or sarcosine (thermal stability points, 44 or 43 °C, respectively). Additionally, the solid triangles in Figure 4 reveal that urea alters the enzyme structure at 35 °C, which is obviously lower than the control denaturation point. This may be due to the fact that the denaturant urea apparently cooperates with the thermal denaturation that might be affecting the catalytic structure of enzyme at 35 °C. It is interesting to note that urea deleterious effects combined with temperature-perturbing actions seem to enhance the thermal denaturation of CT. Counteracting Effects of Methylamines on Urea Deleterious Actions on CT by DLS Measurements. One aim of this work is to elucidate the attenuation effects of methylamines on urea perturbing actions on CT on the basis of the values of dH of CT in methylamine, urea, and their combinations. Initially, to check the compatibility of methylamines and the denaturation of urea, we conducted the DLS measurements for obtaining dH

of CT as a function of methylamine or urea concentrations individually. Figure 5 compares the representative effects of CT in TMAO, betaine, sarcosine, urea, and methylamine mixed with urea at various ratios. The results in Figure 5 explicitly show that the dH values of CT in methylamines, open circles in parts a-c, are not appreciably changed as a function of methylamine concentration (for example, dH values are 12.1 ( 0.09, 12.6 ( 0.15, 14.1 ( 0.30, 14.6 ( 0.10, 15.0 ( 0.26, and 16.1 ( 1.68 nm for 0 (control), 0.5, 1, 1.5, 2, and 3 M TMAO, respectively), indicating that the native structure is not significantly changed with increasing methylamine concentration. Our results are quite corroborated with the DLS studies of Samaddar et al.,58 in which the native glutaminyl-tRNA synthetase (GlnRS) hydrated radius did not increase as a function of protein concentration in the presence of a naturally occurring osmolyte, L-glutamate. The results explicitly indicate that naturally occurring methylamines interact unfavorably with the disulfide bonds of the enzyme, while these do not interfere with the functional activity of the enzyme and these osmolytes preserving the enzyme’s activity. In contrast, the presence of urea


Venkatesu et al.

TABLE 2: DLS Measurements of Hydrodynamic Diameter (dH) for CT in TMAO, Betaine, Sarcosine, and Urea Separately and Mixed at Various Ratios at 25 °C solution

dH (nm)

solution

dH (nm)

buffer 0.5 M TMAO 1 M TMAO 1.5 M TMAO 2 M TMAO 3 M TMAO 0.5 M betaine 1 M betaine 1.5 M betaine 2 M betaine 3 M betaine 0.5 M sarcosine 1 M sarcosine 1.5 M sarcosine 2 M sarcosine 3 M sarcosine 0.5 M urea 1 M urea 2 M urea 3 M urea 4 M urea 5 M urea 6 M ureaa

12.1 ( 0.09 12.6 ( 0.15 14.5 ( 0.30 14.6 ( 0.10 15.0 ( 0.26 16.1 ( 1.68 13.9 ( 0.15 15.4 ( 0.15 16.1 ( 0.56 17.2 ( 0.50 18.9 ( 1.72 15.6 ( 0.02 16.4 ( 0.22 25.5 ( 0.61 30.6 ( 0.61 40.2 ( 1.17 24.3 ( 0.12 32.3 ( 0.65 44.1 ( 1.09 55.5 ( 1.15 66.8 ( 2.25 80.5 ( 4.22 235 ( 12.65

0.5:1 TMAO:urea 1: TMAO:urea 1:2 TMAO:urea 1.5:3 TMAO:urea 1:3 TMAO:urea 1:4 TMAO:urea 1:5 TMAO:urea 2:4 TMAO:urea 0.5:1 betaine:urea 1:1 betaine:urea 1:2 betaine:urea 1:3 betaine:urea 1.5:3 betaine:urea 1:4 betaine:urea 1:5 betaine:urea 2:4 betaine:urea 0.5:1 sarcosine:urea 1:1 sarcosine:urea 1:2 sarcosine:urea 1:3 sarcosine:urea 1.5:3 sarcosine:urea 1:4 sarcosine:urea 1:5 sarcosine:urea 2:4 sarcosine:urea

24.3 ( 0.32 25.6 ( 0.18 35.5 ( 0.52 42.7 ( 0.80 45.3 ( 1.09 54.2 ( 1.05 65.0 ( 1.11 50.9 ( 1.18 26.8 ( 0.48 27.8 ( 0.21 39.2 ( 0.46 48.6 ( 3.76 51.5 ( 0.73 57.0 ( 4.00 68.3 ( 3.64 55.6 ( 0.94 28.5 ( 0.76 30.9 ( 0.89 41.3 ( 0.86 52.1 ( 1.26 53.5 ( 0.79 63.0 ( 1.80 74.5 ( 2.36 66.4 ( 1.08

a

This point is not shown in Figure 5.

TABLE 3: Transition Temperature (Tm), Calorimetric Enthalpy Change (∆H), and Heat Capacity Change (∆Cp) Determined by DSC and Calculated Gibbs Free Energy Changes in Unfolding State at 25 °C for the r-Chymotrypsin in Different Solvent Mediaa sample buffer 0.5 M TMAO 1 M TMAO 2 M TMAO 3 M TMAO 0.5 M betaine 1 M betaine 2 M betaine 3 M betaine 0.5 M sarcosine 1 M sarcosine 2 M sarcosine 3 M sarcosine 1 M urea 2 M urea 3 M urea 4 M urea 5 M urea 6 M urea 0.5:1 TMAO:urea 1:1 TMAO:urea 1:2 TMAO:urea 1:3 TMAO:urea 1:4 TMAO:urea 1:5 TMAO:urea 2:4 TMAO:urea 0.5:1 betaine:urea 1:1 betaine:urea 1:2 betaine:urea 1:3 betaine:urea 1:4 betaine:urea 1:5 betaine:urea 2:4 betaine:urea 0.5:1 sarcosine:urea 1:1 sarcosine:urea 1:2 sarcosine:urea 1:3 sarcosine:urea 1:4 sarcosine:urea 1:5 sarcosine:urea 2:4 sarcosine:urea

Tm ∆H ∆GU ∆Cp (°C) (kJ · mol-1) (kJ · mol-1) (kJ · mol-1. · K-1) 54.3 58.3 61.1 65.4 70.2 57.4 60.3 63.6 68.4 56.2 58.7 62.3 67.1 48.1 45.4 42.0 39.6 37.3 34.6 57.0 61.4 58.4 57.9 57.5 57.1 58.8 56.1 60.5 57.6 57.0 56.1 55.3 57.8 55.5 57.0 55.9 55.4 54.9 54.0 56.4

394 542 705 858 952 505 635 746 854 430 508 640 756 312 255 217 195 101 52 475 612 588 559 549 525 595 450 570 521 491 465 422 561 445 521 452 422 400 386 511

35.0 54.1 75.9 102.0 125.0 49.2 67.0 85.3 108.3 40.4 51.3 70.9 93.4 22.3 16.2 11.6 9.0 3.9 1.6 45.7 66.3 59.0 55.4 53.6 50.8 60.4 42.3 60.3 51.1 47.4 43.8 38.7 55.2 41.0 50.2 42.2 38.7 36.2 34.0 48.3

0.210 0.150 0.124 0.094 0.080 0.163 0.143 0.112 0.091 0.185 0.164 0.130 0.107 0.230 0.265 0.298 0.341 0.381 0.401 0.161 0.170 0.169 0.170 0.177 0.181 0.166 0.171 0.176 0.180 0.183 0.191 0.196 0.173 0.181 0.201 0.184 0.188 0.195 0.208 0.182

a Each value is the average over three measurements. The error in Tm does not exceed 0.1 °C. The estimated relative uncertainties in ∆H, ∆GU, and ∆Cp are around 2% of the reported values.

Figure 6. Differential scanning calorimeter thermograms for CT in 0.05 M Tris-HCl buffer solution, pH 8.20. The inset (a) represents heat flow as a function of temperature and the determination of enthalpy changes by direct integration of the peak in finer scale. The inset (b) displays the prediction of change in heat capacity of unfolding of CT in buffer solution.

significantly increases the dH values of CT with increasing urea concentration in the buffer solution, from 12.1 ( 0.09 to 80.5 ( 4.22 nm in 0 to 5 M urea solutions and 235.0 ( 18.65 nm in 6 M urea solution, revealing that urea is perturbing the functional activity of the enzyme and loss of its functional activity while interacting favorably with the enzyme surface through the cleavage of disulfide bonds of the enzyme. Eventually, to obtain the detailed comparison of counteracting effects of methylamines, we measured dH of CT in the mixtures containing the molar ratio of 1:2 methylamine:urea and varying the urea concentrations (from 1 to 5 M) in the presence of 1 M methylamine. Figure 5a shows that TMAO can strongly offset the actions of urea on CT at the ratio of 1:1 as well as at a 1:2

ratio of TMAO and urea. For example, the dH value of CT in 1 M TMAO is 14.1 ( 0.30 nm and that in 1 M urea is 32.3 ( 0.65 nm. Note that TMAO as combined with urea at 1:1 ratio; the observed dH is 25.6 ( 0.18 nm, considerably lower than the values in the presence of individual 1 M urea. Furthermore, increasing the urea concentration (in the range of 1-5 M urea) in the mixtures with a fixed concentration of 1 M TMAO, the dH values are still smaller than those in TMAO + urea systems and thus clearly show that TMAO counteracts the deleterious effects of urea on the enzyme. Taken together, these results reveal that TMAO protects against favorable changes induced by urea not only at a 1:2 ratio but also at higher urea concentrations in the presence of 1 M TMAO. Apparently, our results indicate that TMAO is a strongly counteracting osmolyte and also reverses the effect of the enzyme structures promoted by urea, due to the highly unfavorable interactions of TMAO with the functional groups of CT. Additionally, It can be seen from Figure 5 (part b or c) that the deleterious effect of urea on



Figure 7. Counteracting effects of methylamine against urea-induced denaturation of CT. The melting point (Tm), which corresponds to the transition of the enzyme to the unfolded state, of CT in methylamines (O) [(a) TMAO, (b) betaine, and (c) sarcosine] and urea (∆) as well as the combination of various ratios of methylamine:urea (b). The point 54.27 °C is the control, which represents the CT transition temperature in buffer solution (the solid line). The dashed lines show the region of methylamine + urea.

CT should be absolutely compensated for by the rest of two methylamines, betaine and sarcosine, at 1:2 methylamine:urea as well as at higher urea concentrations in the presence of 1 M betaine or sarcosine. Figure 5 illustrates the counteracting effects of the methylamine series, indicating that TMAO strongly attenuates the effect, betaine gives moderate offset effect, and sarcosines has little counteracting ability caused by urea on CT. For example, Table 2 shows that the values of dH are 14.1 ( 0.30, 15.4 ( 0.15, 20.5 ( 0.22, and 44.1 ( 1.09 nm in the cases of 1 M TMAO, 1 M betaine, 1 M sarcosine, and 2 M urea, respectively. It is interesting to note that, with methylamine as combined with urea at a 1:2 ratio, the dH value of 2 M urea was decreased from 44.1 ( 1.09 to 35.2 ( 0.52 nm for 1:2 TMAO:urea, 39.2 ( 0.46 for 1:2 betaine:urea, and 41.3 ( 0.86 for 1:2 sarcosine: urea. Thus our results strongly indicate that, in the contribution of methylamine combined with urea, TMAO seems to give stronger counteracting osmolyte than betaine or sarcosine on perturbing effects caused by urea on CT on the basis of DLS measurements. Thermodynamic Protein Stability and Thermal Denaturation. To obtain the compatibility of methylamines and urea perturbing actions on CT, we have performed thermal unfolding

experiments by DSC. Calorimetric heat flow (H) and melting temperature (Tm) of CT in different solvent media were determined from Pyris Software, which was included in the DSC. The enthalpy changes (∆H) were obtained from directly integrating the peak area of DSC thermal histograms. The heat capacity (∆Cp) of CT in various solvent media can be calculated as follows:

∆Cp )

[ ]

60E H Hr m

(2)

where E is the cell calibration coefficient, H is the difference in heat flow between sample and blank, Hr is the heating rate, and m is the sample mass. The quantity 60E/Hr is a constant under a given set of experimental conditions. The heat capacity of unfolding changes is obtained at denaturing temperature Tm. The data analyses of obtaining Tm, ∆H, and ∆Cp are presented in Figure 6. From this figure initially no peaks are visible on the computer screen at all cases and this is quite consistent with our phase behavior analysis of CT via a supercritical antisolvent process59 and CT thermal denaturation in ionic liquids,42 which shows that all transitions


Venkatesu et al.

Figure 8. Counteracting effects of methylamine against urea-induced denaturation of CT. The enthalpy changes (∆H) of unfolding for CT in methylamines (O) [(a) TMAO, (b) betaine, and (c) sarcosine] and urea (∆) as well as the combination of various ratios of methylamine:urea (b). The point 394.37 kJ mol-1 is the control, which is CT in buffer solution (the solid line). The dashed lines show the region of methylamine + urea.

are irreversible. Eventually, we obtained the peaks in finer scale and the analysis was depicted in Figure 6. On the other hand, thermodynamic analysis of the thermal denaturation of CT additionally permitted for the calculations of the Gibbs free energy of unfolding at 25 °C (∆GU), which is a better indication of global protein folding studies, by using the Gibbs-Helmholtz equation60 as follows:

∆GU(T) ) ∆H[1 - (T/Tm)] - ∆Cp[(Tm - T) + T ln(T/Tm)] (3) The results of the thermodynamic parameters of CT in different solvent media are collected in Table 3 and are displayed in Figures 7-10. Counteracting Effects of Methylamines on Urea-Induced Denaturation of CT by DSC Measurements. Virtually, the unfavorable interactions of cosolvents with proteins account for the increase of protein folding, indicating that free energy changes associated with the enthalpy changes increase with increasing concentration of naturally occurring osmolytes, while ∆G and ∆H decrease with increasing concentration of denaturants. This indicates that the interactions are favorable and the denaturants destabilize the proteins.5,8 Figure 7 compares the

change in Tm values, which corresponds to the transition of CT to the unfolded state, as a function of methylamine or urea concentration. It can be seen that TMAO, betaine, or sarcosine rapidly increase Tm values with increasing methylamine concentrations (changing from 54.27 °C in the absence of osmolytes to 70.17, 68.39, and 67.13 °C in the presence of 3 M of TMAO, betaine, and sarcosine, respectively), while urea lowers Tm values with increasing urea concentration (changing from 54.27 °C in the absence of urea to 42.04 and 34.64 °C in the presence of 3 and 6 M urea, respectively). The results explicitly indicate that methylamine increases the transition temperatures and keeps the folding form beyond 67-70 °C, which are obviously higher than the control point (the case without adding any osmolytes). This conclusion is also in good agreement with their behavior of protecting osmolytes. Interestingly, the transition-temperature order of the folding formed by methylamines shows that TMAO is the strongest stabilizer, while betaine is a moderate stabilizer and sacrosine is a weak stabilizer. On the contrary, urea (1 and 6 M urea) depresses the Tm by 6.17 and 19.63 °C, respectively, with respect to the control, which can distinctly be related with its denaturation power. Parts a-c of Figure 7 depict that Tm values of CT in methylamine plus urea mixture (methylamine and urea at molar ratios of 1:1 and 1:2 and the urea concentrations varying in the



Figure 9. Counteracting effects of methylamine against urea-induced denaturation of CT. The Gibbs free energy changes (∆GU) at 25 °C of unfolding for CT in methylamines (O) [(a) TMAO, (b) betaine, and (c) sarcosine] and urea (∆) as well as the combination of various ratios of methylamine:urea (b). The point 34.97 kJ mol-1 is the control, which is CT in buffer solution (the solid line). The dashed lines show the region of methylamine + urea.

presence of 1 M methylamine) rapidly increase and these mixtures have almost the same contribution that the Tm values of methylamine-alone actions on CT, except for the Tm (54.03 °C) of CT in 1:5 sarcosine:urea (Figure 7c), which slightly crossed the control point (54.27 °C). Note that Tm values of CT in 1 M sarcosine and 5 M urea are 58.65 and 37.28 °C (Table 3), respectively. It is worth noting that, with sarcosine as combined with urea at a 1:5 ratio, the transition point (Tm ) 54.03 °C) sharply comes close to the individual effects of 1 M sarcosine. This reveals that sarcosine may also counteract the urea denaturation of CT in 1:5 sarcosine:urea. Thus, our results strongly indicate that the contribution of methylamine combined with urea is thermodynamically the same contribution as that for the methylamine alone on CT. These findings reveal that a methylamine plus urea mixture substantially increases Tm values and offsets the urea-induced denaturation of CT. Furthermore, Figures 8 and 9 show that ∆H and ∆GU values of CT in methylamine at pH ) 8.20 increase with increasing concentration of methylamine, while those values decrease in the presence of urea. In our previous studies,5,21 we also found that the similar trend of the transfer free energies (∆G′tr) of

cyclic dipeptides, which were obtained from solubility measurements, increases linearly with osmolyte concentration while decreasing with increasing concentration of denaturant. The results indicate that methylamine interacts unfavorably with CT and these osmolytes stabilize CT, while these do not interfere with the functional activity of the macromolecules. In other words, urea, in contrast to osmolyte, interacts favorably with protein surfaces, indicating that the disulfide bonds of CT are disturbed and preferential binding with the surface of the enzyme occurs. It emerges from Figures 8 and 9 that the deleterious actions of urea on the unfolding of CT should be apparently compensated for by methylamines at 1:2 methylamine:urea ratio as well as the function of urea concentration (1-5 M) in the presence of 1 M methylamine. However, 1 M sarcosine sharply crosses the control point for CT in 1:5 sarcosine:urea ratio, and the perturbing effect of CT in 5 M urea is balanced by the stabilizing effect of CT in 1 M sarcosine. In fact, ∆H and ∆GU values for CT in 1 M sarcosine (508.19 and 51.25 kJ mol-1, respectively) may be counteracted by the combination of 1:5 sarcosine:urea ratio (385.69 and 33.98 kJ mol-1, respectively) against the perturbing effect of CT in 5 M urea (101.02 and


Venkatesu et al.

Figure 10. Counteracting effects of methylamine against urea-induced denaturation of CT. The heat capacity changes (∆Cp) of unfolding for CT in methylamines (O) [(a) TMAO, (b) betaine, and (c) sarcosine] and urea (∆) as well as the combination of various ratios of methylamine:urea (b). The point 0.2099 kJ mol-1 K-1 is the control, which is CT in buffer solution (the solid line). The dashed lines show the region of methylamine + urea.

3.90 kJ mol-1, respectively) as shown in Table 3. Therefore, this phenomenon is further evidence that methylamine can strongly offset the actions of urea on CT at the ratio of 1:2 as well as CT in the mixtures containing higher urea concentrations (3-5 M) + 1 M methylamine. Additionally, a similar type of behavior was observed in the offset effects of methylamine against the urea actions on CT on the basis of heat capacity changes (Figure 10). These thermodynamic profiles reveal that TMAO is a strong stabilizing and counteracting osmolyte, while betaine and sarcosine are moderate and weak stabilizing and counteracting osmolytes, respectively. Methylamine and Urea Effects on CT and Methylamine Counteracts Urea-Induced Denaturation of CT. To obtain more detailed understanding of the urea effects, compatibility, and counteracting ability of methylamines, we have performed parallel DLS and DSC experiments for CT in methylamine and urea and their mixtures at different ratios at pH 8.20. Both of our results systematically reveal that methylamine interacts unfavorably with the disulfide bonds of CT and the zwitterionic form of methylamine stabilizes the CT, while the stabilizing methylamine tends to be excluded from the enzyme surface, forcing the polypeptide to adopt a compactly folded structure

with a minimum of exposed surface area by water molecules. Methylamine enhances water structure and forms a hydration layerwithwatermolecules.Duringthisperiod,theCys191-Cys220 disulfide bond26 (as mentioned in the Introduction) and a surrounding network of CT also form a hydration layer with water molecules. Apparently, the polypeptide of CT is less able to interact with hydrated water around methylamine. Therefore, there is negative binding between methylamine and the Cys191-Cys220 disulfide, and eventually methylamine was preferentially expelled by the surface binding site of the enzyme. Interestingly, our results are corroborated with a simple statistical mechanics backbone solvation model,8 and the experimental studies,5,21,61-63 in which the protecting osmolytes raise the free energy of the unfolded state, favoring the folded population. In other words, our results are implying that urea interacts more favorably than does water with the disulfide bonds, particularly Cys191-Cys220 disulfide, indicating that the magnitude of hydrophobic effect of catalytic activity decreases.25 The massive penetration of the interior of CT by urea, and it is not surprising that the surface regions of CT are substantially perturbed by urea. The denaturant is considered to act by breaking proteins bonds and accumulate at the surface of the


Figure 11. Schematic depiction of CT (a) preferential hydration and (b) preferential binding in the presence of TMAO and urea, respectively. Moreover, c is the TMAO counteracting effect; i.e., the mixture of TMAO and urea behaves like the individual effect of TMAO.

protein, thus appearing to be bounded, and the protein is noted to be preferentially binding. For urea, binding interactions overcome the stabilizing of excluded methylamine effects and promote unfolding of the protein. Subsequently, this behavior is consistent with the results from molecular dynamics64,65 and experimental studies.21,44-46 Those studies concluded that urea is directly bound to the protein surface. The difference between preferentially binding and preferential hydration is shown schematically in Figure 11. For the sake of clarity in the presentation we have shown only TMAO interactions with CT in this graph. Our results strongly reveal that methylamine protects against favorable changes induced by urea not only at a 1:2 ratio but also at higher urea concentrations in the presence of 1 M methylamine. This conclusion explains that the contribution from methylamine combined with urea in CT is thermodynamically similar to that from the methylamine alone in CT. Therefore, our studies strongly indicate that methylamines are counteracting osmolytes and also reverse the effect on CT structure promoted by urea, due to the highly unfavorable interaction of methylamine with the functional groups of CT.

J. Phys. Chem. B, Vol. 113, No. 15, 2009 5337 It is widely argued that the counteracting ability of the osmolyte arises from the unfavorable interactions between osmolyte + urea and protein, in which the combination of osmolyte + urea is preferentially excluded from the vicinity surroundings of protein.14,19,21,23 There have also been simulation studies19,23 of aqueous solutions that included TMAO, urea, and proteins, which might explain how TMAO counteracts against the urea actions on proteins. Bennion and Daggett23 observed that TMAO enhanced water-water hydrogen bonding both in TMAO-water mixtures and in urea-TMAO-water mixtures. In addition, TMAO also strengthened water-water and water-urea interactions66,67 and led to a decrease in urea-protein hydrogen bonding. In such a situation, TMAO limits the urea denaturing effects. Bennion and Daggett23 clearly depicted these findings that urea was observed to be sandwiched between water molecules, and that formed the hydration layer over the methyl groups of TMAO. Urea is not free to penetrate into the enzyme surface. In such a situation, TMAO limits the urea denaturing effects. This enhancement of solvent structure in hydration shell prevents the initial attack of the protein by water and subsequently urea; further, it protects the protein against the urea actions. The schematic illustration of TMAO counteraction against the deleterious urea actions on CT is also included in Figure 11. The results of this study explicitly delineate that naturally occurring TMAO is a powerful and prevalent, while sarcosine is a least effective, counteracting osmolyte on urea perturbing effects on CT. Analogously, Strambini and Gonnelli,61 very recently, predicted that among the three methylamines TMAO is the most effective and sarcosine the least effective in counteracting the ice perturbing actions on protein. In fact, the extent of stabilization is proportional to the number of methylamine groups directly attached to the nitrogen atom.68 Note that TMAO has three methyl groups directly attached with nitrogen atom,5 thereby inducing the strongest effect in compatibility as well as counteracting effects. The results indicate that these methylamines have comparable effects on counteracting the reversed actions of urea on the protein and also act independently on the protein. Conclusions To study the compatibility and counteracting effects of methylamines such as TMAO, betaine, and sarcosine and the denaturing actions of urea on R-chymotrypsin (CT), we have measured the hydrodynamic diameter and calorimetric data (Tm, ∆H, ∆GU, and ∆Cp) using DLS and DSC, respectively, for CT in various concentrations of TMAO, betaine, sarcosine, and urea, separately and their mixtures containing the molar ratio of 1:2 of methylamine:urea and varying the urea concentrations (from 1 to 5 M) in the presence of 1 M methylamine. Furthermore, to check the methylamine offset effects against the temperature perturbation effect on the enzyme, we also measured the hydrodynamic diameter (dH) values of CT in 1 M methylamine and 1 M urea as a function of temperature through the DLS measurements. Our parallel (DLS and DSC) results reveal that the stabilizing methylamines tend to exclude from the enzyme surface, forcing the polypeptide to adopt a compactly folded structure, while the urea accumulates at the enzyme surface, appearing to be preferentially binding and promoting unfolding of the enzyme. Our findings reveal that methylamine is able to strongly counteract the destabilizing effects of urea on CT in all cases, due to the highly unfavorable interactions between methylamine and CT that overcome the favorable interactions between urea

5338 J. Phys. Chem. B, Vol. 113, No. 15, 2009 and CT. Moreover, our DLS measurements clearly show that methylamine may not only counteract the effects of chemical perturbation of urea on CT but also disrupt protein function and structure by a temperature variation. We have shown in this study that TMAO exerts a powerful stabilizer and most effective counteracting osmolyte, whereas sarcosine is the weak stabilizer and least effective osmolyte to the perturbing urea actions on CT. It is probable that all effective stabilizing solutes will counteract the effects of a chemical perturbant, urea or GdnHCl, a physical variable, and variation of temperature, and there is a need to investigate the effects of variations in solute on a selection of different proteins and models of enzymes. Additionally, the factors affecting folding/unfolding, wider ranges of models, and solutes may facilitate the design of new stabilizing and counteracting studies with protein potential applications in biotechnology. Acknowledgment. Financial support from the National Science Council, Taiwan, through Grant No. NSC 96-2811-E011-003 is gratefully acknowledged. References and Notes (1) Yancey, P. H.; Somero, G. N. Biochem. J. 1979, 183, 317. (2) Yancey, P. H.; Clark, M. E.; Hand, S. C.; Bowlus, R. D.; Somero, G. N. Science 1982, 217, 1214. (3) Record, M. T., Jr.; Courtenay, E. S.; Cayley, D. S.; Guttman, H. J. Trends Biochem. Sci. 1998, 23, 143. (4) Lambert, D.; Draper, D. E. J. Mol. Biol. 2007, 370, 993. (5) Venkatesu, P.; Lee, M. J.; Lin, H. M. J. Phys. Chem B 2007, 111, 9045. (6) Burg, M. B.; Ferraris, J. D.; Dmitrieva, N. I. Physiol. ReV. 2007, 87, 1441. (7) Yancey, P. H. J. Exp. Biol. 2005, 208, 2819. (8) Street, T. O.; Bolen, D. W.; Rose, G. D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13997. (9) Lopes, D. H. J.; Sola-Penna, M. Arch. Biochem. Biophys. 2001, 394, 61. (10) Schellman, J. A. Biophys. J. 2003, 85, 108. (11) Bowlus, R. D.; Somero, G. N. J.Exp. Zool. 1979, 208, 137. (12) Sola-Penna, M.; Lemos, A. P.; Favero-Retto, M. P.; MeyerFernandes, J. R.; Vieyra, A. Z. Naturforsch. C 1995, 50, 114. (13) Sola-Penna, M.; Ferreira-Pereira, A.; Lemos, A. P.; MeyerFernandes, J. R. Eur. J. Biochem. 1997, 248, 24. (14) Lin, T. Y.; Timasheff, S. N. Biochemistry 1994, 33, 12695. (15) Baskakov, I.; Wang, A.; Bolen, D. W. Biophys. J. 1998, 74, 2666. (16) Burg, M. B.; Peters, E. M.; Bohren, K. M.; Gabbay, K. H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6517. (17) Palmer, H. R.; Bedford, J. J.; Leader, J. P.; Smith, R. A. J. J. Biol. Chem. 2000, 275, 27708. (18) Ortiz-Costa, S.; Sorenson, M. M.; Sola-Penna, M. Arch. Biochem. Biophys. 2002, 408, 272. (19) Zou, Q.; Bennion, B. J.; Daggett, V.; Murphy, K. P. J. Am. Chem. Soc. 2002, 124, 1192. (20) Holthauzen, L. M. F.; Bolen, D. W. Protein Sci. 2007, 16, 293. (21) Venkatesu, P.; Lee, M. J.; Lin, H. M. Arch. Biochem. Biophys. 2007, 466, 106. (22) Zancan, P.; Almeida, F. V.; Faber-Barata, J.; Dellias, J. M.; SolaPenna, M. Arch. Biochem. Biophys. 2007, 467, 275. (23) Bennion, B. J.; Daggett, V. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6433. (24) Branden, C.; Tooze, J. Introduction to Protein Structure; Garland Publishing, Taylor & Francis Group: New York, 1999. (25) Creighton, T. E. Proteins, Structures and Molecular Properties, W. H. Freeman, New York, 1993.

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Osmolyte Counteracts Urea-Induced Denaturation of Î±-Chymotrypsin

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