General Mechanism of Osmolytes' Influence on Protein Stability

Article pubs.acs.org/JPCB

General Mechanism of Osmolytes’ Influence on Protein Stability Irrespective of the Type of Osmolyte Cosolvent Aneta Panuszko,* Piotr Bruździak, Emilia Kaczkowska, and Janusz Stangret Department of Physical Chemistry, Chemical Faculty, Gdańsk University of Technology, Narutowicza 11/12, 80-233 Gdańsk, Poland S Supporting Information *

ABSTRACT: The stability of proteins in an aqueous solution can be modified by the presence of osmolytes. The hydration sphere of stabilizing osmolytes is strikingly similar to the enhanced hydration sphere of a protein. This similarity leads to an increase in the protein stability. Moreover, the hydration sphere of destabilizing osmolytes is significantly different. These solutes generate in their surroundings so-called “structurally different water”. The addition of such osmolytes causes “dissolution” of the specific protein hydration sphere and destabilizes its folded form. No relationship is seen between the stabilizing/destabilizing properties of osmolytes and their structure-making/-breaking influence on water. Furthermore, their accumulation at the protein surface or their exclusion does not determine the osmolytes’ effect on protein stability. An explanation to the osmolytes’ stabilizing/ destabilizing influence originates in the similarity of water properties in osmolytes and protein solutions. The spectral infrared characteristic of water in an osmolyte solution allowed us to develop practical criteria for classifying solutes as stabilizing or destabilizing agents.

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INTRODUCTION The native structure of a protein is usually very sensitive to any change in its environmental properties such as temperature, pressure, humidity, salinity, and so forth. Their variations can cause unwanted changes in the activity of the protein or in its secondary and tertiary structures. Living organisms have developed a few strategies to counteract environmental stress. One of them, quite universal to many kingdoms of living organisms, is the accumulation of small organic molecules called osmolytes. These small and soluble organic or inorganic compounds can be highly concentrated (1−2 mol/L), without any significant influence on the metabolism of a cell.1−3 Although the name osmolytes can refer to any solutes influencing the osmotic pressure of a solution, it is customary to use that name only for selected groups of compounds. Apart from their segregation on the basis of their chemical origin, osmolytes can be divided into two groups: stabilizing (many amino acids, some amines and their derivatives, sugars, etc.) and destabilizing (mainly urea and its derivatives, guanidinium chloride), with regard to proteins. The stabilizing or destabilizing properties seem to be universal and independent of the chemical characteristics of the protein. There is no model that completely explains the influence of osmolytes on macromolecule stability at the molecular level. One of the most popular theories is the theory of preferential exclusion.4−17 According to this hypothesis, stabilizing osmolytes are preferentially excluded from the protein surface and favor preferential hydration of the protein. On the other © 2016 American Chemical Society

hand, destabilizing osmolytes (e.g., urea) possess the ability to interact with the surface and directly facilitate the unfolding process. However, the hypothesis does not provide the details on the molecular basis of stabilization and may not always be true.18 Another popular theory states that osmolytes act indirectly by altering the water structure (weakening or strengthening the hydrogen bond).19−25 The water structure hypothesis predicts that stabilizing/destabilizing properties of osmolytes are determined by their structure-making/-breaking influence on water.19−25 Despite the popularity of the water structure hypothesis, there is a lack of data that determine an osmolyte’s effect on the water structure or the data are in conflict. Therefore, some authors have suggested15,16,26−28 that there is no clear relationship between an osmolyte’s influence on the water structure and its effect on protein stability. In our opinion, these two hypotheses are not mutually exclusive. They rather give a picture of the same problem from two different, yet complementary, points of view. In our previous papers, hydration of stabilizing osmolytes (trimethylamine N-oxide,29 glycine and its derivatives,30,31 and amino acids32) was investigated. Interesting conclusions were drawn concerning the role of hydration water in maintaining the thermal stability of lysozyme in solutions of these stabilizing osmolytes.18,31 Using the same experimental method, only urea hydration was determined so far.29 To fully understand the Received: October 6, 2016 Published: October 7, 2016 11159

DOI: 10.1021/acs.jpcb.6b10119 J. Phys. Chem. B 2016, 120, 11159−11169

Article

The Journal of Physical Chemistry B

water−osmolyte systems were collected according to the procedure presented in ref 18. The Nicolet 8700 spectrometer (Thermo Electron Co.), with a single-reflection diamond crystal Golden Gate ATR accessory (Specac), was used to collect all spectra. The temperature during the measurements was kept at 25 ± 0.1 °C using an electronic temperature controller (Specac). Each spectrum was an average of 512 independent scans (resolution 2 cm−1). The “turbo” mode of the spectrometer’s EverGlo source provided a better signal-tonoise ratio. The spectrometer and ATR accessory were purged with dry nitrogen. All spectra were smoothed with the 8-point Savitzky−Golay filter. The residual between raw and smoothed spectra exhibited no spectral features; thus, no information was lost in the smoothing step. The procedure for isolation of the spectra of the affected osmolytes is described in detail in refs 42 and 43 and in the Supporting Information (Section S1). It is based on the difference spectra method described in Section S3.2. Because of the large amount of spectral data, most of the results are given in the Supporting Information section. DSC. The CSC 6300 nano-DSC III calorimeter (CSC Corp.) with capillary platinum cells (0.299 mL) was used to collect calorimetric data. The scanning rate was kept at 1 K min−1. The temperature range for each experiment was fixed at 25−95 °C. DSCRun software (CSC Corp.) was used to run calorimetric experiments. The data was analyzed with Matlab-based scripts. All thermograms and other fitted parameters of protein denaturation models (one-step reversible, one-step irreversible, and Lumry−Eyring model) are available in the Supporting Information section. Because of the problems with baseline fitting, all fitted parameters should be considered with caution. It was impossible to measure the influence of TMU on the protein thermal stability by means of the DSC calorimeter because of the gelation process occurring in the TMU−protein solution. Such a gel could not be precisely placed in a thin capillary cell of the DSC calorimeter. Methods Used to Study Osmolyte−Water Systems. FTIR Spectroscopy. Infrared spectra of aqueous solutions of urea derivatives (resolution of 4 cm−1, in the range of 500−5000 cm−1, 128 independent scans) were recorded on the Nicolet 8700 spectrometer (Thermo Electron Corporation). Dry nitrogen was used to purge the spectrometer’s interior. A liquid transmission cell (model A145, Bruker Optics) was used, with two CaF2 windows separated by Teflon spacers and a path length of 0.0289 mm (determined interferometrically). A thermocouple was placed in the cell to monitor the sample temperature and to adjust it, if necessary (25.0 ± 0.1 °C), with an electronic temperature controller. Analysis of HDO Spectral Data. The following computer software was used to collect and analyze the FTIR spectra: OMNIC (Thermo Electron Corporation), GRAMS/32 (Galactic Industries Corporation, Salem, NH), and RAZOR (Spectrum Square Associates, Inc., Ithaca, NY) run under GRAMS/32. The difference spectra method was applied to isolate the solute-affected HDO spectrum. The method is described in detail in refs 42−46, and the most important information is included in Section S3.2. An assumption is made that water in solution can be grouped into two distinctive populations: bulk water (identical to pure water) and solute-affected water. Both have characteristic spectral features and, according to the Lambert−Beer law, should be additive. The solute-affected water spectrum gives valuable information about the energetic

destabilizing influence of denaturing osmolytes on a biomolecule, other small organic compounds must be taken into consideration. Here, we propose a group of urea alkyl derivatives: N-methylurea (NMU), N-ethylurea (NEU), N-nbutylurea (NBU), N,N′-dimethylurea (DMU), N,N′-diethylurea (DEU), and N,N,N′,N′-tetramethylurea (TMU). These compounds are not well characterized in the context of both hydration33−35 and their influence on protein thermal stability.36−41 In this article, a set of complementary experimental and computational results have been used. Attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy has allowed us to determine whether any strong interactions between these urea derivatives and protein (hen egg white (HEW) lysozyme) were present in aqueous solution. Differential scanning calorimetry (DSC) was used to describe the stability of proteins in urea solutions. By means of FTIR spectroscopy, the hydration of urea derivatives was investigated. The method of HDO isotope dilution in H2O solutions allowed us to isolate the OD band of HDO, which was narrower and less complex than the OH band of H2O and was practically free from intramolecular and intermolecular couplings between oscillators, which served as a probe of the water structure around selected compounds. As a result, a full description of the FTIR-derived hydration of urea derivatives is presented and correlations are made showing a direct link between the protein thermal stability and the properties of water affected by these denaturing agents. Our methodology gives a larger picture of protein−water− osmolyte interactions and may bring us closer to the full mechanism of protein−solute mutual influence. In the case of many stabilizing or denaturing osmolytes, the water structure is enhanced and false conclusions can be made concerning the water-mediated osmolytes’ influence on protein stability. The nature of these changes is much more complex, but a few characteristic features of water affected by osmolytes can be found that explain the observed effects in protein−osmolyte solutions, as shown in this article.

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EXPERIMENTAL SECTION Chemicals and Solutions. NMU (98%; Aldrich), NEU (97%; Aldrich), NBU (99%; Aldrich), DMU (98%; Sigma), DEU (97%; Aldrich), TMU (99%; Fluka), and D2O (isotopic purity 99.9%; Aldrich) were used without further purification. The HEW lysozyme purchased from Fluka was prepared as in ref 18. Depending on the experimental methods, the final concentrations of lysozyme in solutions were as follows: ca. 1.6 mg mL−1 (DSC studies) and 0−200 mg mL−1 (ATR-FTIR studies on an osmolyte’s influence on protein stability in aqueous solutions; the osmolytes’ concentrations were kept constant, whereas the protein concentrations were varied). In the first case, all solutions were prepared in 20 mM phosphate buffer, pH 6.5, as in ref 31, for better comparison with our previous results and to diminish the influence of pH variations on protein stability. However, in the second type of experiment, focused on the interaction of osmolytes with the protein, the solutions were not buffered, for better clarity on the observed interactions. Sample and reference solutions for FTIR measurements of HDO spectra were prepared as described in the Supporting Information (Section S3.1). Methods Used to Study Lysozyme−Osmolyte−Water Systems. ATR-FTIR Spectroscopy. All spectra of lysozyme− 11160


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Figure 1. (a−g) ATR-FTIR spectra of urea and its derivatives (solutes): bulk reference spectrum of a solute extrapolated to 0 mol/kg (black lines); spectra of the solute affected by its own presence in a solution, that is, resulting from an increase in its concentration (red lines); spectra of the solute affected by the presence of lysozyme (blue lines) interpolated to a common solute molality of 1 mol/kg. (h) Spectroscopic preferential interaction coefficients in solute−lysozyme systems.

spectra of osmolytes in aqueous solutions (without protein). In the case of TMU and urea, changes can be considered as significant on the basis of the carbonyl region of the ATR-FTIR spectra. Generally, the shape of the spectra of urea and its derivatives in aqueous solutions suggests that these solutes are able to interact with other molecules of solutes, either by creation of weak hydrogen bonds or through a dipolar attraction or both. In most cases, the protein in such a solution increases the effect corresponding to solute−solute interactions, as observed in solute−water systems. This conclusion can be drawn from the comparison of solute−solute affected spectra (red spectra in Figure 1) and solute−protein affected spectra (blue spectra in the same figure), which are quite similar. In the case of dialkyl urea derivatives and tetramethylurea, an association of alkyl substituents to the hydrophobic residues of

state of the hydrogen bonds of water and intermolecular distances of hydrating water molecules. However, the method does not provide any information about the geometry of hydrogen bonds and dynamics of water molecules, which is a limitation of the method. Details concerning the interpretation of the solute-affected water spectrum have been described in Section S3.5.

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RESULTS ATR-FTIR Investigation of the Protein−Osmolyte Interaction. Changes in the band shape of the isolated osmolytes’ spectra (1700−1500 cm−1) in the presence of lysozyme are generally quite small (Figure 1). The lysozymeaffected spectra of urea derivatives are similar to the bulk spectra of these solutes in most cases, that is, the isolated 11161


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interior and rather directly promote thermal unfolding at high temperatures. The hypothesis of a different influence mechanism is supported by the denaturation model analysis (Tables S2−S4). The DSC denaturation curve of lysozyme in the presence of urea, NMU, NEU, DMU, and DEU can be easily fitted with the Lumry−Eyring model (Figures S15−S26), whereas the same curve corresponding to the presence of NBU can be fitted with only the one-step irreversible model (Figure S27). The experimental error of calorimetric enthalpy of denaturation is difficult to estimate; thus, the values of calorimetric enthalpy of denaturation and its derivatives must be obtained with care. However, the sign of the slope of ΔHcal versus m dependence is clearly negative in the case of all urea derivatives (Figure 2b) and positive in the case of glycine and

the protein surface can be expected. In such a case, the carbonyl group may experience some destabilizing interactions with adjacent polypeptide residues. We can cautiously state that such interactions occur in the studied systems on the basis of the appearance of an additional sub-band in the high-wavenumber region of the carbonyl band of these solutes, with a maximum above 1650 cm−1 (Figure 1f,g). Deeper insight into the changes in the band shape of urea derivatives caused by the presence of protein suggests that, in most cases, these interactions are not specific in nature or that the energy of interactions with the protein is comparable to that of water molecules. The number of lysozyme-affected osmolyte molecules are generally similar in the case of each urea derivative (ca. 20−30), suggesting a limited number of possible interaction centers existing on the protein surface. Relatively, NBU exhibits the largest number of affected molecules per mole of the osmolyte and, as a consequence, the largest Ps coefficient (Figure 1h). However, the corresponding affected spectra are only weakly distorted compared to the bulk unaffected NBU spectrum. Thus, a high number of solute molecules being affected by the protein does not mean that these interactions are strong. However, in each case, the Ps coefficient tends to drop to 1 at a higher concentration, suggesting that at higher concentrations the composition of the solution near the protein surface is similar to that of the bulk solution (Figure 1h). Such a situation is similar to the previously described case of the proline− lysozyme system and is significantly different from the case of the TMAO−lysozyme system, in which TMAO has been excluded from the protein surrounding (Ps < 1).18 Thus, we can conclude that urea and its derivatives are accumulated in the protein surrounding. DSC Experiments: Protein Thermal Stability. A dTm/dC parameter was used to compare the influence of an osmolyte on the thermal stability of a protein.31 The dTm/dC parameters for all osmolytes are presented in Table 1. Not surprisingly, all Table 1. Change of Lysozyme’s Melting Temperature in the Presence of Urea and Its Derivatives solute

dTm/dCa

(dTm/dC)/Nb

U NMU NEU NBU DMU DEU

−3.2 −5.5 −7.5 −28.6 −6.8 −12.9

−0.6 −3.1 −3.1 −9.2 −2.5 −4.0

Figure 2. Calorimetric enthalpy of lysozyme denaturation in the presence of (a) stabilizing osmolytes31 and (b) urea and its derivatives. The legends contain information on the slope of ΔHcal vs m dependence.

its methyl derivatives (Figure 2a). This indicates that urea and its derivatives lower the denaturation enthalpy, in contrast to the group of stabilizing osmolytes.31 FTIR Investigation of Water Structure. A detailed analysis of the FTIR spectra of the denaturing osmolytes’ solution series and the interpretation of osmolyte-affected water spectra have been described in Sections S3.3−S3.5. Figure 3a shows the osmolyte-affected HDO spectra (without the ND contribution) and the bulk water spectrum (for comparison; all spectra were scaled to a common maximum). The oxygen− oxygen distance distribution function, P(ROO) (Figure S3b), in water is calculated according to the equation shown in Section S3.5. The band parameters for the affected HDO bands together with those for the bulk HDO bands are presented in Table 2, along with intermolecular oxygen−oxygen distances, ROO. To visualize the difference in intermolecular distances, ΔP(ROO), the following procedure was used: From the distance

a

Change in the protein denaturation temperature per mole of solute (°C dm3 mol−1). bThe change attributable to one mole of water molecules affected by the solute (°C dm3 mol−1 molH2O−1).

dTm/dC parameters are negative and their values clearly depend on the size and number of alkyl substituents. These results are consistent with those from previous reports.36−41 The denaturation temperature in the presence of glycine and its methyl derivatives, by contrast, was not strictly dependent on the number of methyl groups.31,47 Each carbon atom in the alkyl substituents is responsible for reduction of the denaturation temperature by ca. −2.4 °C. However, longer substituents, such as n-butyl, do not follow this rule. NBU has a greater influence (dTm/dC = −28.6 K/mol) on Tm than does DEU (dTm/dC = −12.9 K/mol), with the same number of carbon atoms in the alkyl groups (four). Most probably, a longer alkyl chain can interact with the hydrophobic protein 11162


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Figure 4. (a) Differences between the interatomic oxygen−oxygen distance distribution functions of the affected water, Pa(ROO), and bulk water, Pb(ROO), calculated as ΔP(ROO) = Pa(ROO) − Pb(ROO) for urea and its alkyl derivatives. The vertical dashed line shows the position of the most probable distance for bulk water at 2.83 Å. (b) Illustration of the method for calculating the area corresponding to the population of weak hydrogen bonds of water molecules, which have been replaced by stronger ones in the hydration sphere of the solutes.

Figure 3. (a) Solute-affected HDO spectra in the OD stretching region for urea and its alkyl derivatives, together with the bulk HDO spectrum (black, dashed). The spectra have been scaled to the same maximum absorption value for better comparison. (b) Interatomic oxygen−oxygen distance distribution function derived from the HDO spectra affected by urea and alkyl urea derivatives, along with the bulk HDO (black, dashed) distance distribution curve.

the population of weak hydrogen bonds of bulk water in the hydration sphere of osmolytes, which have been replaced by stronger ones. These are shown in Table 3. The α parameter increases in the following order: urea < TMU < NEU < NBU < DEU < DMU < NMU. The decrease in the population of weak hydrogen bonds in water is characteristic of hydrophobic solutes and can be understood as a sign of hydrophobic hydration. A similar effect was observed for the tertbutylammonium45 and tetraethylammonium48 cations studied as models of hydrophobic hydration. In the case of urea and its derivatives, the α parameter comprises the contribution of alkyl substituents as well as amino and carbonyl groups in the hydration shell. To estimate the contribution of only alkyl groups, we used the urea

distribution function of water affected by osmolyte, Pa(ROO), the distribution function for bulk water, Pb(ROO), was subtracted. The results of the subtraction are shown in Figure 4a for each studied osmolyte. Influence of the Alkyl Group on the Surrounding Water Molecules. It is possible to quantitatively describe the vanishing of weak water−water hydrogen bonds on the basis of the above-mentioned ΔP(ROO) distribution function (Figure 4a). The vanishing of weak hydrogen bonds can be calculated as the area of the difference curve under the ROO axis, α (as shown in Figure 4b). These values reflect the contributions of

Table 2. Parameters of HDO Bands Affected by Denaturing Osmolytes and Those of Bulk HDO Bands along with Their Respective Intermolecular Oxygen−Oxygen Distancesa bulk Uj NMU NEU NBU DMU DEU TMU

νoODc

Nb

solute 5.0 1.8 2.4 3.1 2.7 3.2 3.8

± ± ± ± ± ± ±

0.5 0.5 0.5 0.5 0.5 0.5 0.5

2509 2509 2511 2505 2507 2503 2501 2511

± ± ± ± ± ± ± ±

νgODd 2 2 2 2 2 2 2 2

2505 2503 2484 2492 2490 2486 2488 2494

± ± ± ± ± ± ± ±

fwhhe 2 2 2 2 2 2 2 2

162 159 153 144 141 147 146 160

± ± ± ± ± ± ± ±

If 4 4 4 4 4 4 4 4

10 096 8482 7627 9882 7336 11 148 11 195 12 169

RoOOg 2.828 2.826 2.823 2.823 2.821 2.821 2.821 2.821

± ± ± ± ± ± ± ±

0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003

RgOOh

ΔEgH−Bi

± ± ± ± ± ± ± ±

−0.4 −4.2 −2.6 −3.0 −3.8 −3.4 −2.2

2.843 2.841 2.821 2.828 2.826 2.823 2.823 2.831

0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003

a ROO errors have been estimated on the basis of HDO bands position errors. bAffected number. cBand position at maximum (cm−1). dBand position at gravity center (cm−1). efwhh (cm−1). fIntegrated intensity (dm3 mol−1 cm−1). gThe most probable O···O distance (Å). hMean O···O distance (Å). i The average change in the energy of hydrogen bonds for one mol of the affected water (kJ). jData are taken from ref 29.

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osmolytes investigated in our present and recent research (glycine and its derivatives, other amino acids, and urea and its derivatives), the smallest substituent, the single methyl group, has the strongest structure-making properties (Table S7 and Figure S32).

Table 3. Contributions of a Vanishing Weak Hydrogen Bonds Population in the Hydration Shell of Solutes and Solvent-Accessible Surface of Urea and Its Alkyl Derivatives solute

αa

Δαb

βc

Δα/β × 10−3 d

Ue NMU NEU NBU DMU DEU TMU

0.021 0.124 0.096 0.104 0.117 0.107 0.068

0.103 0.075 0.083 0.096 0.086 0.047

38.81 70.94 126.91 79.36 140.31 127.49

2.668 1.062 0.657 1.209 0.617 0.372

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DISCUSSION Effect of Destabilizing Osmolytes on the Water Structure. The shift of the most probable (RoOO) and mean (RgOO) distances toward lower values (i.e., shorter oxygen− oxygen distances), in comparison to those in bulk water (Table 2), indicates that water−water hydrogen bonds are stronger in the surroundings of denaturing osmolytes. From comparison of the position of the center of gravity band values, νgOD (related to the mean energy of water hydrogen bonds), for destabilizing osmolytes and bulk water (Table 2), it is clear that water affected by these osmolytes forms on average stronger H-bonds than those in pure water. The average change in the water− water hydrogen bond energy (ΔEgH−B; see Section S3.5) increases in the following order: bulk water < urea < TMU < NEU < NBU < DEU < DMU < NMU, just like that for the α parameter. Thus, NMU appears to be the best hydrogen bond enhancer among these solutes. The differences in intermolecular distances, ΔP(ROO) (Figure 4a), clearly show that the population of very weak water−water hydrogen bonds (ROO > 2.9 Å) in the presence of all destabilizing osmolytes decreases. All osmolytes increase the population of mean energy hydrogen bonds (i.e. the population of water−water hydrogen bonds is only slightly longer than or equal to the most probable distance in bulk water, value of RoOO = 2.83 Å in Table 2). This effect increases with the length of an alkyl substituent, both in the case of monosubstituted (NMU < NEU < NBU) and disubstituted urea derivatives (DMU < DEU). At the same time, the probability of occurrence of short (ROO < 2.8 Å) water−water hydrogen bonds increases. The maximum of the ΔP(ROO) function is close to 2.75 Å in each case, which corresponds roughly to the distance typical to water in the ice phase (2.76 Å). The substitution of the hydrogen atom of urea with one methyl group increases the ice-like water population in a solution. However, the increase in the substituent length reduces the effect. The same can be observed in the case of disubstituted urea derivatives. In the final analysis, it is evident that urea and its derivatives, despite their destabilizing properties, with respect to proteins, enhance the water structure in their nearest vicinity, although urea influences it rather weakly. It should be pointed out that when we write “enhanced” it should be understood as enhanced with respect to the mean hydrogen bond energy of water molecules and to the mean intermolecular oxygen−oxygen distance in water. There are conflicting reports in the literature concerning the structure-making/-breaking effect of urea and its alkyl derivatives. Some studies are in agreement with our results and have shown that urea and its alkyl derivatives possess “structure-making” properties,34 whereas other research works have indicated that they act as water “structure breakers”.33,35 Differences in the Stabilizing and Destabilizing Osmolytes’ Influence on Water Structure. Our results obtained for denaturants and previously published results for stabilizers29,32 demonstrate that both the groups of osmolytes tend to enhance the water structure, and simple hydration parameters (like νgOD or ΔEgH−B and RgOO from Tables 2 and S6) seem to categorize all of them as “structure-making”

a

The parameter that reflects the contributions of vanishing weak hydrogen bonds of water molecules. The α parameter was calculated as an area (see Figure 4b and the text for details). bThe α parameter for the alkyl derivatives of urea relative to urea (α − αu), determining the contribution of the vanishing weak hydrogen bonds of water molecules around the alkyl group of urea. cThe solvent-accessible surface for the alkyl group of urea (Å2). dThe contribution of the vanishing population of weak hydrogen bonds of water molecules per unit surface of the alkyl group (Å−2). eThe value for urea was calculated on the basis of the difference in the intermolecular distance distribution function, ΔP(ROO), shown in ref 29.

molecule as a model, which takes into account interactions of water molecules with the amino and carbonyl groups. The contribution of vanishing weak hydrogen bonds of water molecules around the alkyl chain, Δα, was obtained by subtracting the value of parameter α obtained for urea from the value of parameter α for each of the other alkyl derivatives of urea. Then, we calculated the contributions of the vanishing population of weak hydrogen bonds of water molecules per unit surface of the alkyl chain, Δα/β. The solvent-accessible surfaces of alkyl chain β have been calculated by the grid method described by Bodor et al.,49 with the atomic radii of Gavezzotti50 implemented in the HyperChem 8.0.3 package. All data used to calculate Δα/β are included in Table 3. The dependence of Δα/β on the number of carbon atoms in the alkyl substituents of the urea derivatives (Figure 5) clearly shows that a single methyl group has the greatest enhancing influence on the water structure, and the more the carbon atoms present in the urea derivative, the weaker the influence it evokes in the surrounding water structure. In each group of

Figure 5. Contributions of the vanishing population of weak hydrogen bonds of water molecules per unit surface of the alkyl group (see Table 3) as a function of the number of C atoms in the alkyl chains for urea derivatives. 11164


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osmolytes according to their influence on water structure (Figure 6b). A simple graph visualizing the way the values of ΔP(ROO) at 2.83 Å differentiate osmolytes is shown in Figure S34a. According to this criterion, osmolytes can be divided into two classes: denaturants (with ΔP(ROO) at 2.83 Å > 0) and stabilizers (with ΔP(ROO) at 2.83 Å < 0). It should be noted that similar relationships were obtained for the ΔP(ROO) value at the mean ROO for bulk water (2.84 Å). It turns out that the full-width half-height (fwhh) of the OD band can be another helpful criterion differentiating the influence of osmolytes on water structure in aqueous osmolyte systems. Moreover, this parameter can be easily derived from affected water spectra. The fwhh of the OD band of affected water takes values higher than that of the OD band for bulk water (162 cm−1) in the case of stabilizing osmolytes and lower than that of the OD band for bulk water in the case of destabilizing ones (Figure S34b). Thus, the fwhh parameter can be used as a rough estimate of a compound’s ability to stabilize or destabilize a protein. The discussion clearly shows that an organic osmolyte can be classified as denaturing or stabilizing agent according to its hydration water properties in solution. Two experimental parameters are given: (1) values of ΔP(ROO) at the most probable water−water distance (2.83 Å) and (2) the fwhh, which allows us to predict the denaturing or stabilizing properties of an osmolyte. Both of these parameters are highly correlated (Figure 7).

solutes. This clearly indicated that the making/breaking water structure does not seem to be a measure of the stabilizing or destabilizing ability of osmolytes. The correlation between ΔEgH−B and α, shown in Figure S33, describes the ability of an osmolyte to enhance the water structure; however, it does not differentiate them. To generalize and confront the influence of both stabilizing and destabilizing osmolytes on the water structure, the average differences in the ROO distribution functions, ΔP(ROO), have been calculated separately for stabilizing and destabilizing osmolytes and are shown in Figure 6a, with ΔP(ROO)

Figure 6. (a) Averaged ΔP(ROO) calculated separately for all denaturing osmolytes (red line, based on Figure 4a) and all stabilizers (blue line, refs 29 and 32), and ΔP(ROO) corresponding to the HEW lysozyme in water (black line, from ref 51). (b) The result of subtraction of averaged ΔP(ROO) functions obtained for denaturing (D) and stabilizing (S) osmolytes (from (a)). The dashed vertical line denotes the most probable oxygen−oxygen distance in bulk water (2.83 Å).

Figure 7. Correlation of ΔP(ROO) values at the most probable water− water distance (2.83 Å) and the fwhh of the OD band (values are taken from Table 2 for denaturing osmolytes and from Table S6 for stabilizing osmolytes). The dashed horizontal line denotes the fwhh of the OD band for bulk water (162 cm−1).

corresponding to the lysozyme−water system. In solutions of stabilizing osmolytes, the population of weak and average hydrogen bonds decrease, whereas stronger hydrogen bonds are favored. The average difference, ΔP(ROO), for the stabilizing osmolytes is very similar to the difference corresponding to the water affected in the hydration sphere of the lysozyme molecule. In the case of denaturants, a decrease in the weaker and increase in the stronger hydrogen bonds populations are likewise observed. However, the water around destabilizing osmolytes exhibits different properties from the water in the hydration shell of stabilizing osmolytes and in the protein hydration shell. The hydration sphere of destabilizers exhibits an increased population of mean energy of hydrogen bonds, with respect to bulk water; therefore, we call it as structurally different water. Consequently, the population of mean energy hydrogen bonds, with respect to bulk water (measured at the most probable ROO for bulk water, i.e., 2.83 Å), can be considered as one of the indicators differentiating

Correlations of the calorimetric parameter (dTm/dC) with values of ΔP(ROO) at 2.83 Å and with fwhh are shown in Figure 8a−c, respectively. However, because of small differences in the values calculated for urea derivatives, these correlations must be carried out with care. Nonetheless, the destabilizing properties of these compounds seem to be governed by the properties of water population characterized by average hydrogen bonds with respect to bulk water. Thus, it is the structurally different water that is affected by urea derivatives that influences the melting temperature of lysozyme the most. The long alkyl substituent of NBU causes a significantly larger change in the protein melting temperature, probably due to possible hydrophobic interactions of the substituent with the hydrophobic interior of the protein. However, its influence on the water structure is very similar to the influence of other 11165


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water affected by these solutes directly influences the protein and is the key factor in the prospective use of other protein stability modifiers. Mechanism of Water-Mediated Protein Stabilization/ Destabilization by Osmolytes. Before we start to describe the water-based mechanism of protein stabilization by osmolytes, we have to depict the process from the thermodynamic point of view. Protein denaturation is an endothermic process; therefore, according to the van’t Hoff equation, the equilibrium constant for this reaction at a given temperature decreases with an increase in the denaturation enthalpy. In other words, the equilibrium constant decreases with an increase in enthalpic stabilization of the protein-folded form relative to that of the unfolded form. Thus, the decrease in the denaturation enthalpy, observed in the case of urea and its derivatives, causes an increase in the equilibrium constant and, in effect, favors the denatured state in relation to the native one, that is, decreases the Gibb’s free energy of the denaturation. The opposite effect is seen in the case of stabilizing osmolytes, fo which the denaturation enthalpy increases in the presence of osmolytes, that is, the native state of the protein was stabilized against the denatured one. The fwhh of the infrared stretching band corresponding to water affected by stabilizing osmolytes is greater than that for bulk water, and especially than that for water affected by destabilizing osmolytes. In simple terms, this parameter is a measure of the structural (including ROO distances) and energetic (EH−B) distribution states of water molecules. A greater fwhh of the band results in a wider distribution, and thus higher entropy and more thermodynamically advantageous states of water molecules. Similarly, a wider distribution is characteristic of protein hydration.51 Therefore, the addition of stabilizing osmolyte to the aqueous solution of the protein exerts not only an enthalpic but also an entropic stabilizing effect the native state of the protein, moving the unfolding reaction equilibrium in the direction of the folded form. Opposite effects are caused by destabilizing osmolytes. The spectrum of protein-affected water51 is composed of three main contributions, that is, of water affected by carboxyl groups, basic groups, and the backbone. The contribution of lysozyme hydration water molecules corresponding to sidechain carboxyl groups is small (ca. 7.8−9.4% of surface area),18,51 whereas the hydration of basic groups is similar to that of bulk water.52 Thus, these two contributions can be omitted in further analysis of water affected by osmolytes. However, the shape of HDO affected by the protein is highly similar to the shape of water affected by N-methylacetamide,51 which serves as a good model of the protein backbone. Therefore, the energetic−structural state of the protein hydration water, which differs from that of bulk water, is mainly determined by the hydration of the backbone. Such a hydration provides the native structure of the protein. Bulk water will tend to “dissolve” the specific protein hydration sphere (or in other words it will try to make it more similar to bulk water) by a dynamic averaging of the states of water molecules. To explain the mechanism of impact of the stabilizing and destabilizing osmolytes on the thermal stability of the protein, we must take into account the effect of cooperative hydrogen bonds in water. The hydrogen-bonded network of specified groups of water molecules surrounded by water molecules with a strong hydrogen bond network is enhanced. Consequently, it is weakened when it is surrounded by a weak network of these bonds. In the case of stabilizing

Figure 8. (a) Correlation of dTm/dC and values of ΔP(ROO) at the most probable water−water distance (2.83 Å). (b) dTm/dC parameter corrected for one molecule of affected water (N) vs values of ΔP(ROO) at the most probable water−water distance (2.83 Å). (c) dTm/dC parameter corrected for one molecule of affected water (N) vs the fwhh of the OD band. The dashed horizontal line denotes the fwhh of the OD band for bulk water (162 cm−1).

derivatives. This indicates that other mechanisms of protein destabilization also take place. We suspect that in higher temperatures, the hydrophobic n-butyl substituent can penetrate through the protein interior, causing quicker unfolding. A different destabilization mechanism is validated by the DSC result, which indicates that the protein denaturation in this case follows one-step irreversible denaturation rather than the two-step mechanism exhibited in the case of other urea derivatives. The hydration properties are directly connected to the thermal stability of lysozyme in solutions. This implies that 11166


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leads to increased protein stability in solutions of the stabilizing osmolytes. These osmolytes shift the equilibrium constant of protein denaturation to that of the native state of the protein, leading to enthalpic and entropic stabilization of the folded state relative to the unfolded one. Destabilizing osmolytes exert an opposite effect on protein stability. In the presence of denaturing osmolytes, with “structurally different” properties of hydration water, the enhanced hydration sphere of the protein seems to be “dissolved”, with a differently modified hydration water of denaturants. The incompatibility of these hydration spheres causes weakening of the hydrophobic effect, and as a consequence, the stability of the protein decreases. There is no clear relationship between osmolytes−protein interactions, that is, the accumulation of osmolytes at the protein surface or their exclusion, and the stability of the protein. In addition, our data show that there is no direct correlation between the water structure-making or “structurebreaking” properties of osmolytes and their effect on protein stability. However, the way the osmolyte was hydrated is the key factor that determines protein stability. To be precise, the similarity, or a lack of such a similarity, between the hydration sphere of the osmolyte and that of the protein is the most important factor.

osmolytes, we observe a high similarity of their hydration sphere relative to the protein hydration sphere, as measured by the oxygen−oxygen distance distribution function of the water molecules. This similarity includes a reduction in the population of water−water hydrogen bonds at the most probable distance for bulk water (2.83 Å) and an increase in the shorter distances, which correspond to stronger hydrogen bonds. According to the effect of cooperative hydrogen bonding in water, the protein hydration sphere is enhanced relative to the situation in which an osmolyte is not present in the solution. In the case of destabilizing osmolytes, we observe a significant difference in the characteristics of their hydration spheres relative to the protein hydration sphere. The hydration sphere of the destabilizers is characterized by the presence of the population of water−water hydrogen bonds at the most probable distance in bulk water (2.83 Å). The increased number of water molecules, with the energy of hydrogen bonds being weaker than the most probable distance for the protein hydration sphere, in accordance with the effect of hydrogen bond cooperativity, reduces the energy of the hydrogen bond in the protein hydration sphere. In this sense, the addition of such osmolytes causes a dissolution of the specific protein hydration sphere and destabilizes its folded form to a greater extent than that in bulk water. This way, the protein can lose its hydration shell and become more vulnerable to a temperature change. Current theories assume that denaturing osmolytes interact with the protein, whereas stabilizing osmolytes are excluded from the protein surface. Our FTIR investigation of lysozyme in aqueous solutions of denaturing osmolytes indicates that these compounds actually accumulate at the protein surface, whereas stabilizers are either accumulated (proline)18 or excluded (TMAO)9,18 from the protein surface. Therefore, accumulation or exclusion is not the main determinant of the effect of osmolytes on protein stability. Moreover, all analyzed osmolytes (stabilizers and destabilizers) enhance the water structure in their nearest vicinity; therefore, “breaking” or “making” of the water structure is also not an indicator of protein stability. The main factor determining the stabilizing/ destabilizing influence of an osmolyte on a protein is the ability of the osmolyte to modify water molecules in a compatible/ noncompatible way, with respect to the structural and dynamic properties of water in the protein hydration sphere.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b10119. Determination of the protein-affected osmolyte spectra, ATR spectroscopy results, DSC results, details concerning the determination and interpretation of the soluteaffected water spectra, and FTIR spectral analysis results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +48 58 3471238. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Marta Cichosz for her assistance in experimental measurements of the lysozyme− osmolyte−water systems. This research was partially supported by the PL-Grid Infrastructure and TASK Computational Center. This work was supported by the Polish National Science Center (NCN) based on decision No. DEC-2013/11/ B/NZ1/02258.

SUMMARY AND CONCLUSIONS All analyzed stabilizing and destabilizing osmolytes enhance the water structure. However, spectroscopic and calorimetric studies allow us to find differences in the influence on water structure, crucial for the explanation of the influence of osmolytes on protein stability. We propose new criteria differentiating the influence of osmolytes on the water structure that are compatible with their stabilizing or destabilizing properties: (a) the fwhh of the OD band with respect to that of bulk water, which is higher in the case of stabilizing osmolytes and lower for destabilizing ones and (b) the population of water molecules with the mean energy of hydrogen bonds with respect to bulk water, which is negative for stabilizing osmolytes and positive for the destabilizing ones. Furthermore, we propose a rough mechanism of protein stabilization/destabilization by osmolytes. The hydration sphere of the protein, mainly determined by the hydration of the protein backbone, is strikingly similar to the hydration sphere of the stabilizing osmolytes; in other words, they are compatible. The compatibility of enhanced hydration spheres

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