Influence of Osmolytes and Denaturants on the Structure and Enzyme

To whom correspondence should be addressed. (P.V.) E-mail: [email protected]; [email protected]. Telephone: +91-11-27666646-142. Fax:...
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J. Phys. Chem. B 2010, 114, 1471–1478

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Influence of Osmolytes and Denaturants on the Structure and Enzyme Activity of r-Chymotrypsin Pankaj Attri,† Pannuru Venkatesu,*,† and Ming-Jer Lee‡ Department of Chemistry, UniVersity of Delhi, Delhi - 110 007, India, and Department of Chemical Engineering, National Taiwan UniVersity of Science and Technology, 43 Keelung Road, Section 4, Taipei 106-07, Taiwan ReceiVed: September 25, 2009; ReVised Manuscript ReceiVed: NoVember 13, 2009

Enzymes are very sensitive and highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of cosolvents, the fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways, and alternatively, enzymes can be stabilized or destabilized. To understand the contribution of osmolytes and denaturants on the stabilization, related to the associated structural changes and enzyme activity of R-chymotrypsin (CT), we have monitored differential scanning calorimeter (DSC), circular dichroism (CD), enzyme activity, and gel electrophoresis as a function of osmolyte or denaturant concentration. The present investigation compares the compatibility of osmolytes and deleterious effects of denaturants on the structure, function, and enzyme activity of CT. This comparison has provided new important insight on the contribution of cosolvent effects on protein folding/unfolding, enzyme activity, and understanding of protein-solvent interactions. Our DSC results reveal that the enthalpy change (∆H) and Gibbs free energy of change (∆Gu) of CT in osmolyte (trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, and sucrose) increase linearly as osmolyte concentration increases, while those values decrease sharply in the presence of denaturants (urea and guanidine hydrochloride (GdnHCl)). The modifications in the secondary structure of this β/β protein, as quantified by the CD spectra, showed reasonable enhancement for β-strands in the presence of the osmolytes as compared to buffer, which contributes to its stabilization power. Evidently, we observed that naturally occurring osmolytes have a dominant contribution to the stabilization of CT while not enhancing its enzyme activity. In contrast, our results revealed that the denaturants enhanced the surface of the enzyme by binding to the surface of CT, which leads to zero enzyme activity. Introduction The detailed structural information and function of enzymes in cosolvents are crucial for understanding their metabolic role and their use as industrial biocatalysts. 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.1 Bacterial serine proteinases share the chymotrypsin-like bilobal β-barrel structure, but they are more distantly related due to their shorter sequences and structural differences in surface loops.2-6 Proteolytic enzyme R-chymotrypsin (CT), which has potential for use in industrial applications, is composed of two juxtaposed β-barrel domains, with catalytic residue bridging and disulfide bridges that join the three polypeptide chains. Apparently, the crystalline structure of CT has a total of five disulfide bonds2 (Protein Data Bank, ID code 2CHA). Out of five disulfides, two (Cys1-Cys122 and Cys42Cys58) are close to the enzymatic active site, which is a catalytic center, and one (Cys191-Cys220) is close to the well-known surface binding site, which is located in the vicinity of the 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). * To whom correspondence should be addressed. E-mail: venkatesup@ hotmail.com; [email protected]. Telephone: +91-11-27666646142. Fax: +91-11-2766 6605. † University of Delhi. ‡ National Taiwan University of Science and Technology.

Protein folding plays a central and vital role in the properties of biological macromolecules in solutions, and this protein folding poses tremendous challenges in both modern biological studies and biopharmaceutical contexts. The stability of a globular protein, in general, is the result of a balance between the intramolecular interactions of protein functional groups and their interactions with the cosolvent particles.7 Cosolvents are widely used in the studies of protein folding, protein processes, and interactions that stabilize/destabilize the protein structures. The enzyme surface is initially responsible for the interaction with the environment of the cosolvent particles that contributes to the protein folding/unfolding. A molecular description of the interaction of cosolvents with functional groups of enzymes is lacking, due to the complex structures of folded proteins. Virtually, the native conformation of the globular proteins and enzymes under external osmotic stresses such as dehydration, temperature variations, variable pH, freezing, high salinity, and internal stress such as high concentrations of protein denaturants can be stabilized by the osmotically active solutes, which are small organic molecules, termed as naturally occurring osmolytes (or osmoprotectants).8-11 In other words, these osmolytes, referred to as compatible or protective osmolytes, tend to stabilize the protein structure, without altering macromolecular structure and function.12,13 The stabilization of compact native structures reveals typically results of preferential exclusion of osmolytes from the vicinity of the macromolecule surface. Energetically, unfavorable interactions can exist between osmolyte and hydration surfaces of the protein.2,10,14 The

10.1021/jp9092332  2010 American Chemical Society Published on Web 01/04/2010

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exclusion of osmolytes from the proximity of the protein surfaces inevitably means the inclusion of water in the surface ofproteins,whichisnaturallytermedapreferentialhydration.10,11,15-22 Classical chemical denaturants such as urea and guanidine hydrochloride (designated GdnHCl) are considered to act by breaking protein hydrogen bonds and interact preferentially with the protein surface, thus appearing to be bounded, and the protein is noted to be preferentially binding. Ultimately, this leads to denaturation of the macromolecule. These denaturants can influence not only the protein unfolding but also the ensemble of the native structure.10,11,23-27 The naturally occurring osmolyte shifts the folding equilibrium from the partially unfolded state toward the native state while denaturants push the equilibrium toward the denaturation state.13,28 Obviously, a complete understanding of the protein folding/unfolding and an elucidation of the function, structures, and activity in the diverse enzymes require thermodynamic folding properties, CD analysis, enzyme activity, and gel electrophoresis that provide a quantitative description of the effects of both osmolytes and denaturants. In the present study, the osmolyte and denaturants are chosen as cosolvents, which play a central role in the stabilization of protein structure. CT is one of the valuable biological substances for understanding the mechanism of protein folding or unfolding with the addition of cosolvents.29 A large number of developing different strategies have been dedicated to elucidating the molecular events of CT stability by several researchers.30-37 In addition, a great deal of work has been done by various research groups using various techniques38-42 to understand the changes in structure and unfolding of CT by the addition of urea and guanidinium salts. Nevertheless, understanding of the structurefunction relationship is still one of the challenging tasks, and the comparison of a molecule depiction of the molecular events of osmolytes or denaturants with CT in its native folded state is lacking and numerous issues are unresolved on the folding of CT and its enzyme activity in the presence of cosolvents. Very recently, we have systematically showed the effects of methylamines on CT43 to understand the thermal stability as well as the thermal denaturation of urea actions on CT by hydrodynamic diameter (dH) and folding thermodynamic properties (Tm, ∆H, ∆Gu, and ∆Cp) with dynamic light scattering (DLS) and differential scanning calorimeter (DSC), respectively. From our previous studies, we discovered that the protecting effect of methylamines is very weak with increasing temperature.43 Nonetheless, there has been no systematic documentation of the influence of osmolytes on the native state of CT to understand the thermal stability as well as the thermal denaturation of denaturant action on CT. In the light of these considerations and to gain further insight into the mechanistic basis of thermal stability, denaturation, and enzyme activity of CT in the presence of the osmolytes (TMAO, sarcosine, betaine, proline, and sucrose) and denaturants (urea and GdnHCl) in this study, we have explored the thermodynamic folding properties, CD spectra analysis, activity studies, and gel electrophoresis as a function of cosolvent concentration. We have also 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 (Tm), enthalpy changes (∆H), and heat capacity changes (∆Cp). 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, betaine, proline, and

Attri et al. sucrose and GdnHCl were purchase from Sigma Chemical Co.. Sarcosine was purchased from Fluka Biochemical Co. (Switzerland), 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 using a Mettler Toledo balance with a precision of 0.0001 g. 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 absence of cosolvent at various concentrations at 25 °C for 4 h to attain complete equilibrium. All samples were prepared at 15 mg/mL enzyme concentration in various concentrations of cosolvent (depending on the solubility of CT) for 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 a syringe before performing the measurements. Differential Scanning Calorimeter (DSC). A differential scanning calorimeter DSC 2920 (TA Instruments, New Castle, DE) was used to measure the Tm values for CT in the different liquid media. A certain amount of bubble-free solution was placed into a 0.1 mL DSC sample cell while a 0.1 mL reference cell was filled with an appropriate blank sample of the same solvent media without CT, and then both cells were 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 up to 95 °C with a scanning rate of 1.0 °C /min. All DSC measurements were carried out in triplicate. Calorimetric enthalpy change (∆H) and heat capacity change (∆Cp) were determined from thermograms by using the Universal Analysis software (TA Instrument), which is included with the DSC instrument. The uncertainties of temperature and heat flow readings are (0.02 °C and (0.1%, respectively. To obtain accurate results, the instrument was calibrated with pure water and sapphire. Each value is the average over three measurements. The error in Tm does not exceed 0.1 °C. The estimated relative uncertainties in ∆H, ∆Cp, and ∆Gu are around 2% of the reported values. Circular Dichroism Spectroscopy. Circular dichroism (CD) spectroscopy is a well-known method for the analysis of macromolecules and is often used for understanding the structure and interaction of protein in solution. CD spectroscopic studies were performed using a PiStar-180 spectrophotometer (Applied Photophysics, U.K.) equipped with a Peltier system for temperature control. CD calibration was performed using (1S)-(+)10-camphorsulfonic acid (Aldrich, Milwaukee, WI), which exhibits a 34.5 M/cm molar extinction coefficient at 285 nm and 2.36 M/cm molar ellipticity (θ) at 295 nm. The sample was preequilibrated at the desired temperature for 15 min, and the scan speed was fixed for adaptative sampling (error F 0.01) with a response time of 1 s and 1 nm bandwidth. The secondary and tertiary structures of CT were monitored by using far-UV (190-240 nm) (0.1 cm path length cuvette) and near-UV (250-300 nm) (1.0 cm path length cuvette) spectra, respectively. The CT concentration was 1 mg/mL, and each spectrum was collected by averaging six spectra. Each sample spectrum was obtained by subtracting the appropriate blank media without CT from the experimental enzyme spectrum. The percentages of secondary structures were calculated by using online CD analysis, k2d, ftp://ftp.bork.embl.de/pub/software/andrade, which provides several curve analyzing algorithms.44 r-Chymotrypsin Activity. The activity of CT was measured by the method of Erlanger et al.45 using 0.1 mM Suc-Ala-Ala-

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Pro-Phe-p-nitroanilide (SAPNA) in 50 mM Tris-HCl, pH 8.2, 20 mM CaCl2 as a substrate solution. Substrate solution of about 100 mL was introduced in a beaker which already contained 63 mg of SAPNA and 10 mL of buffer, and this solution was gently stirred for 3 h at room temperature. By this method, we prepared stock solution for obtaining the CT activity in various concentrations (depending on the CT solubility in cosolvent) of cosolvents. Activity was measured by using a UV-vis spectrophotometer (Shimadzu, UV-1601, Tokyo, Japan) at 410 nm and for 3 min. Chymotrypsin activity units were expressed as the change in absorbance per minute per milligram of protein. Specific activity of CT per milliliter of sample was calculated via the following equation.

(Abs410 /min) × 1000 × mL of reaction mixture activity units ) extinction coefficient of chromogen × mg of protein in reaction mixture

(1) The molar extinction coefficient of para-nitroanalidine liberated from chromogens of SAPNA is 8800. Molecular Study of CT in Various Osmolytes and Denaturants. Separation of proteins in the enzyme extracts was carried out by 12% SDS-PAGE.46,47 The CT sample (15 mg/ mL) was introduced into each well, and electrophoresis was performed (30 mA) on a vertical dual mini gel electrophoresis device (Hoefer SE-260, Amersham Pharmacia) at room temperature. The enzyme sample with osmolyte or denaturant was incubated in SDS-PAGE substrate at 50 °C. The gel was then washed with distilled water, stained with 0.1% Coomassie brilliant blue (CBB R-250), and incubated in methanol/acetic acid/water (40:10:40) for 2 h. Destaining was performed with the same solution without CBB R-250 for 1 h. Clear protein bands were observed. The gels were documented with a calibrated densitometer (GS-800, Bio-Rad, CA 94547) with the help of Quantity one - 4.5.1 software. Results and Discussion Thermodynamic Enzyme Stability and Thermal Denaturation. Thermodynamic information is an essential element in the dissection of the structural basis of biological function. The fundamental thermodynamic forces that underlie the structure and function of molecules are the essence of chemical technology. Specifically, the Gibbs free energy change (∆G) is useful to describe the global protein folding studies, whereas the enthalpy change (∆H), heat capacity change (∆Cp), and transition temperature (Tm) are useful to understand the protein stability in terms of the noncovalent forces of the different structural states. Moreover, these properties can provide a more detailed understanding of protein stability, since these thermodynamic parameters can be related more directly to the structure of the protein and interactional parts of the solvent effect. From our DSC analysis, initially no peaks are visible on the computer screen in all cases and this is quite consistent with CT thermal denaturation in ionic liquids,37 our phase behavior analysis of CT via a supercritical antisolvent process,48 and our very recent studies,43 which shows that all transitions are irreversible. Eventually, we obtained the peaks in finer scale, for which the analysis was depicted in Figure 6 of ref 43. In other words, thermodynamic analysis of the thermal denaturation of CT additionally permitted us calculations of the Gibbs free energy of unfolding at 25 °C (∆Gu), which is a better indication

of global protein folding, by using the Gibbs-Helmholtz equation43,49 as follows,

∆Gu(T) ) ∆H[1 - (T/Tm)] - ∆CP[(Tm - T) + T ln(T/Tm)]

(2)

The heat flow for the unfolding of CT was studied by DSC in the presence and absence of osmolytes and denaturants in TrisHCl buffer of pH 8.2. The results of the thermodynamic folding parameters of CT in different solvent media are graphically displayed in Figure 1. The results in Figure 1a distinctly distinguish the change of Tm, values, which correspond to the transition of CT to the unfolded state, as a function of cosolvent concentration. It can be seen that osmolytes rapidly increase Tm values with increasing osmolyte concentration (changing from 54.3 °C in the absence of osmolytes to 65.4, 64.8, 63.6, 62.3, and 61.7 °C in the presence of 2 M TMAO, proline, betaine, sarcosine, and sucrose, respectively) while the denaturant abruptly lowers Tm values with increasing denaturant concentration (changing from 54.3 °C in the absence of denaturant to 39.6 and 20.9 °C in the presence of 4 M of urea and GdnHCl, respectively). Through these effects, we have explicitly found that the osmolyte increases the transition temperatures and keeps the folding form beyond 67-75 °C, which is obviously higher than the control point (absence of osmolyte). These observations evidently reveal that the CT structure of the folding state is not altered by the addition of any osmolyte. This finding supports most strongly our earlier work implicating the peptide backbone unit and other amino acid residues are stoutly in the mechanism events of osmolyte-induced stabilization of cyclic dipeptides.10,11 The stability abilities of the native state of CT varied from osmolyte to osmolyte; therefore, the efficiency of stabilizing effects follows the trend TMAO > proline > betaine > sarcosine > sucrose. Interestingly, the transition temperature order of the folding formed by the osmolytes shows that TMAO is the strongest stabilizer, while proline, betaine, or sarcosine is a moderate stabilizer and sucrose is a weak stabilizer. Looking at the data of unfolding of denaturants (Figure 1a), it is obvious that GdnHCl is a more effect denaturant than urea on perturbing the structure of CT. This observation is quite consistent with our previous result11 and also the effects of denaturants on proteins by the molecular transfer model.50 These observed results reveal that there are large variations in thermal stability of CT as the concentrations of osmolyte and denaturant are increased. This conclusion is also in good agreement with their behavior of protecting osmolytes for macromolecule.9,43 It emerges from Figure 1b and c that the ∆H and ∆Gu values of CT in osmolyte at pH ) 8.20 significantly increase linearly as osmolyte concentration increases, while those values decrease sharply in the presence of urea as well as GdnHCl. In our previous studies,10,11 we also found that the similar trend for the transfer free energies (∆Gtr′) of cyclic dipeptides increases linearly as osmolyte concentration increases while those values decrease with increasing concentration of denaturant. The results indicate that osmolyte interacts unfavorably with the surface of CT and these osmolytes stabilize the folded native structure of CT, while these do not interfere with the functional groups of CT. As the concentration of osmolytes increases, the stabilities of CT also linearly increase. In other words, denaturant, in contrast to osmolyte, interacts favorably with the surface of CT, indicating disruption of the disulfide bonds of CT and preferential binding with the surface of the enzyme.

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Figure 1. Influence of cosolvents on the structure of CT from the thermodynamic enzyme stability and thermal denaturation. The melting point (Tm), which corresponds to the transition of the CT to the unfolded state (A), the enthalpy change (∆H) of unfolding (B), the Gibbs free energy changes (∆Gu) at 25 °C of unfolding (C), and the heat capacity change (∆Cp) of unfolding (D) for CT in the presence of TMAO (open circles), proline (open triangles), betaine (open squares), sarcosine (closed circles), sucrose (closed triangles), urea (closed squares), and GdnHCl (crosses). Solid lines show smoothness of the folding thermodynamic properties.

The origin of the contrasting behavior of ∆CP values for CT in cosolvents reveals that the osmolyte slightly decreases the ∆CP values as the osmolyte concentration raises, whereas denaturant sharply increases with increasing concentration of denaturant (see Figure 1d). These phenomena support our previous work, which delineates that ∆CP values decrease as methylamine concentration increases.43 Interestingly, our results of ∆CP are corroborated with results of Lin et al.,51 in which they observed that ∆CP values are decreased for the folding structure of the Fyn SH3 domain in the presence of TMAO. Preliminary explanations of these effects are that the osmolyte does not significantly alter the folding state structure of CT and also the strength of its hydrophobic structure. Apparently, the increment of the unfolded ∆CP values for CT, due to denaturant, are quite consistent with those of previous studies that denaturant increases the ∆CP values as the denaturant concentration increases.52 It is clear that the denaturation heat capacity increment is caused primarily by transfer of the internal nonpolar groups to water upon protein unfolding. On the other hand, denaturant expands the surface of CT and essentially ruptures disulfide bonds that contribute the heat capacity effect of the increase in the disruption of the native structure of CT. This ultimately leads to a deleterious effect on CT structure with the addition of denaturant. Circular Dichroism Analysis. To obtain the mechanism of events of the osmolytes’ role in enhancement of the stability and the denaturants’ disruption of the CT structure, we further studied CD spectrum analysis. The far-UV CD spectra of the enzyme indicate that the CT has a particular secondary structure

Figure 2. Variation in the far-UV CD spectra analysis of the CT in the presence of control (black), 2 M TMAO (red), 2 M sarcosine (cyan), 2 M sucrose (magenta), 2 M urea (purple), and 2 M GdnHCl (orange) at 30 °C. However, for the sake and clarity of presentation, we have not shown the CD spectra for betaine and proline due to very high absorption.

in each of these cosolvents. The CD spectrum of CT in buffer has no positive band, whereas TMAO and sarcosine show the positive band. Proline and betaine have also very high positive absorption (data not shown here) (Figure 2). Many research groups34,35,37 did not use Tris-buffer for measuring the far-UV CD spectrum (190-240 nm wavelengths) due to its high polarization and high absorption. To solve this problem, we have changed our buffer system to water at pH 8.2. Eventually, there is no variation in CD spectra analysis. The far-UV CD spectra

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TABLE 1: Secondary Structure Composition of r-Chymotrypsin Determined from Far-UV CD Spectra in Different Solvent Media at 30 °C sample

R-helix

β-helix

random

total

buffer TMAO proline betaine sarcosine sucrose urea GdnHCl

0.08 0.02 0.00 0.02 0.02 0.11 0.10 0.13

0.45 0.51 0.00 0.51 0.51 0.64 0.43 0.41

0.47 0.47 1.00 0.47 0.47 0.25 0.46 0.45

1 1 1 1 1 1 1 1

data help us to estimate β-structure using K2d software, and these results are shown in Table 1. The observed results are in agreement with those obtained by other researchers.30,34 In Table 1, TMAO, betaine, sarcosine, and sucrose show clear improvement in the β-structure. This indicates that osmolytes are stabilizing additives by virtue of the fact that they maintain both the solvophobic interactions essential for the native structure and for preserving of the water shell around the CT structure. The drop-off β-structure clearly showed the ability of urea and GdnHCl to ruin the compact and flexible native conformation of CT. The near-UV CD spectra (250-300 nm) were also monitored in order to obtain information on the tertiary structure, which particularly arises from the protein aromatic residues of the enzyme in all the assayed media, and the results are illustrated in Figures 3 and 4. The CD spectrum of CT in the 250-300 nm region is a composite of disulfide nσ*, tyrosyl Lb, and tryptophanyl La and Lb contributions. The Lb transition is extremely sensitive; however, the La transition is less sensitive to conformational change.53,54 This spectral region is dominated by the contribution of aromatic residues (e.g., Trp, Tyr) and disulfide bridges, while the intensity of the 250-300 nm bands are affected by local conformational changes around these chromatophores. Apparently, these results are corroborated by the experimental studies of Kudryashova and co-workers,30 in which CT in buffer medium showed maxima at 254, 289, and 296 nm that are best assigned to Trp residues (see Figure 4). Urea and GdnHCl induced unfolding of the R-subunit of Trp synthase, giving the observed changes in CD at 286 nm (Figure 3). Consequently, the near-UV CD spectra of CT show small differences in intensity obtained for TMAO, betaine, sarcosine,

Figure 4. Near-UV CD spectra analysis of CT in control (black), 2 M TMAO (red), 2 M proline (green), 2 M betaine (blue), 2 M sarcosine (cyan), and 2 M sucrose (magenta) at 30 °C.

Figure 5. Influence of cosolvents on enzyme activity of CT. Relative specific activity of R-chymotrypsin (%) in buffer, 3 M TMAO, 3 M proline, 3 M betaine, 2 M sarcosine, 2 M sucrose, 4 M urea, and 3 M GdnHCl.

Figure 6. Molecular weight marker (M; Bio-Rad, low range) for CT in buffer (D0), 2 M sarcosine (D1), 4 M urea (D2), 3 M proline (D3), 2 M TMAO (D4), 3 M sucrose (D5), 3 M GdnHCl (D6), and 3 M betaine (D7).

Figure 3. Variation in the near-UV CD spectra for CT in the presence of control (black), 2 M urea (purple), and 2 M GdnHCl (orange) at 30 °C.

proline, and sucrose at ∼256, 266, and 278 nm (see Figure 4), which could again be attributed to a compact conformation, in which the internal aromatic residues remain in the hydrophobic core of the CT at a reduced distance. Our aforementioned experimental results concluded that the osmolytes protect the native folded structure of CT while denaturants perturb the native structure of CT.

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TABLE 2: Activity of r-Chymotrypsin by Using 0.1 mM Suc-Ala-Ala-Pro-Phe-p-Nitroanilide (SAPNA) in 50 mM Tris-HCl, pH 8.20 sample

A410 nm/min

CT concentration (mg/mL)

CT in reaction mixture (mg/10 µL)

specific activity (unity/mg)

relative specific activity of CT (%)

buffer 3 M TMAO 3 M proline 3 M betaine 3 M sarcosine 2 M sucrose 4 M urea 3 M GdnHCl

0.6652 0.7102 0.1759 0.5679 0.3245 1.1085 0.0012 0.0017

0.2553 0.5914 0.5574 0.5531 0.5617 0.6765 0.3063 0.2808

0.0025 0.0059 0.0055 0.0055 0.0056 0.0067 0.0030 0.0028

22.5008 ( 0.0012 10.3696 ( 0.0020 2.7252 ( 0.0009 8.8661 ( 0.0016 4.9893 ( 0.0025 14.1493 ( 0.0006 -0.0338 ( 0.0018 -0.0522 ( 0.0010

100.0 46.0 12.1 39.4 22.1 62.8 0.0 0.0

SCHEME 1: Schematic Depiction of CT Preferential Hydration and Preferential Binding in the Presence of TMAO and GdnHCl, Respectivelya

a In preferential binding, denaturant focuses on hydrogen bonding with CT and probably interacts favorably with the enzyme surface, whereas osmolyte is excluded from the CT surface due to steric repulsions from water and obviously unfavorable interactions are occurring between osmolyte and CT surface.

r-Chymotrypsin Activity. To ascertain whether the osmolytes or denaturants cause any alternation in the enzyme activity of the folding transition state of CT, we further performed enzyme activity experiments. Very few studies are available in the literature55 related to CT activity. Therefore, we tested the CT activity in the presence of cosolvent by using a UV-vis spectrophotometer. The obtained results are collected in Table 2 and are displayed in Figure 5. It is quite clear from this figure that urea and GdnHCl have zero enzyme activity as CT is in the unfolded state. Our previous results show that, among all the osmolytes, TMAO is a powerful stabilizer and the most compatible of osmolytes, whereas our activity results reveal that sucrose has eminent activity rather than the rest of the osmolytes (Figure 5). This phenomenon explains that chymotrypsin activity is not significantly enhanced by any osmolytes. The lack of change in CT activity under similar conditions indicates that osmolyte-induced folding is not specific. Our results are consistent with the experimental results of TMAO-induced enzyme activity55 that the protein conformation induced by osmolytes is a natural one and not simply forced, nonspecific. Molecular Study of CT in the Presence of Osmolytes/ Denaturants. In order to study the binding capability of cosolvents to CT structure, we used gel electrophoresis. From

gel electrophoresis, we predicted the molecular weight of CT in the presence of different cosolvents. The results are summarized in Figure 6. Our results reveal that CT bands are completely absent in the case of urea and GdnHCl, that leads to complete denaturation of CT native structure. Further, we observed two bands for CT with molecular weights of 25.89 and 17.37 kDa in sarcosine and indicate that the CT does not hold sarcosine properly. In TMAO, proline, sucrose, and betaine single bands were observed with molecular weights of 31.77, 25.75, 27.79, and 21.20 kDa, respectively. These values are larger than that of buffer (20.78 kDa), which concludes that osmolytes show preferential hydration in the vicinity of CT. TMAO showed the highest molecular weight because of its capability of holding the protein intact more than the rest of osmolytes do. Therefore, this phenomenon further shows that osmolyte can strongly protect the folded state of CT, whereas denaturant perturbs the native structure of enzyme. These studies contribute to the osmolyte-induced preferential hydration analysis which states that osmolytes help in the formation of a compact and flexible nativelike species. Our parallel results systematically reveal that osmolyte interacts unfavorably with the disulfide bonds of CT while the stabilizing osmolyte tends to be excluded from the enzyme surface and forces the polypeptide to adopt a compactly folded

Influence of Osmolytes and Denaturants on CT structure with a minimum of exposed surface area by water molecules. On the other hand, osmolyte enhances water structure and forms a hydration layer with water molecules. During this period, the Cys191-Cys220 disulfide bond3,43 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 the osmolyte. Therefore, there is negative binding between the osmolyte and the Cys191-Cys220 disulfide, and eventually the osmolyte was preferentially expelled by the surface of the CT, which causes the to osmolyte stabilize the native structure of CT. Interestingly, our results are corroborated with a simple statistical mechanics backbone solvation model,13 as well as the molecular transfer model50 and our previous experimental studies10,11,43,56 in which the protecting osmolytes raise the free energy of the unfolded state, favoring to the folded population. On the other hand, our results imply that denaturant interacts more strongly than does water with the disulfide bonds, particularly Cys191-Cys220 disulfide, indicating that the magnitude of hydrophobic effect of catalytic activity decreases.2 With the massive penetration of the interior of CT by denaturant, it is not surprising that the surface regions of CT are substantially perturbed by either urea or GdnHCl. The denaturant is considered to act by breaking proteins bonds and accumulate at the surface of the protein, thus appearing to be bounded, and the protein is noted to be preferentially binding. Consequently, this behavior is quite consistent with the results from molecular dynamics57-59 as well as our previous unfolding studies.10,11,43,56 The difference between preferential binding and preferential hydration is schematically revealed in Scheme 1. For the sake of clarity in presentation, we show only TMAO and GdnHCl interactions with CT in this scheme. Conclusions Our parallel (DSC, CD, enzyme activity, and gel electrophoresis) results reveal that the stabilizing osmolytes tend to be excluded from the enzyme surface, forcing the polypeptide to adopt a compactly folded structure, while the urea or GdnHCl accumulates at the enzyme surface, appearing to be preferentially binding and promoting unfolding of the enzyme. According to our experimental studies, we showed that the osmolytes interact with chymotrypsin in a hierarchical way but do not enhance its enzyme activity. Furthermore, our results reveal that TMAO is a powerful stabilizer and the most effective compatible osmolyte whereas sucrose is a weak stabilizer and the least protective osmolyte. As sucrose is a weak stabilizer, however, it shows highest enzyme activity compared with the other osmolytes. The structure and function of CT are strongly influenced by the cosolvent through interaction between the vicinity of CT surface and environment of the cosolvents. Our systematic experimental results explicitly reveal that the preferential interactions occur between the surface of CT and neighboring particles of cosolvents through exclusion of osmolyte and inclusion of denaturant. Additionally, the factors affecting folding/unfolding in the presence of cosolvents and wider ranges of models and solutes may facilitate the design of new stabilizing and compatible for studies with protein potential applications in biotechnology and biophysical chemistry. Acknowledgment. This project was funded by a Department of Science and Technology (DST, New Delhi, India) grant (SR/ SI/PC-54/2008). We highly acknowledge Mr. Kameshwar Sharma and Mr. Sandeep Garg, University of Delhi for help in doing enzyme activity and gel electrophoresis and also Ad-

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