Hydration-Mediated Effects of Saccharide Stereochemistry on Protein

Chapter 9

Hydration-Mediated Effects of Saccharide Stereochemistry on Protein Heat Stability Downloaded by UNIV OF FLORIDA on November 16, 2017 | http://pubs.acs.org Publication Date (Web): November 2, 2017 | doi: 10.1021/bk-2017-1257.ch009

Renata Kisiliak and Yoav D. Livney* Biotechnology & Food Engineering, Technion, Israel Institute of Technology, Haifa, 3200000, Israel *E-mail: [email protected]

Thermal stability of globular proteins is important in biotechnological, food and pharmaceutical applications. Sugars provide significant protection against thermal denaturation of globular proteins, but the mechanisms of this protection are still incompletely understood, particularly the role of sugar stereochemistry in its effectiveness as a protein stabilizer. To shed new light on this important problem, we systematically studied isomeric sugars within groups of mono- and di-saccharides, and investigated the effect of stereochemistry on sugar hydration, and its impact on protein stability. No binding was found between the sugars and the protein, supporting our hypothesis that sugars affect proteins mainly indirectly via the water. Protein denaturation temperature increased with increasing sugar concentration in the following order of efficacy: Monosaccharides: galactose > glucose > mannose; Disaccharides: trehalose > cellobiose > maltose. Our main finding is that the extent of thermal protection conferred to the protein correlates with the hydration number (nh) of the sugar within each group of isomers.

Introduction Low molecular-weight carbohydrates (also called osmolytes) are known to confer protective effect against deterioration of biological molecules in living © 2017 American Chemical Society Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by UNIV OF FLORIDA on November 16, 2017 | http://pubs.acs.org Publication Date (Web): November 2, 2017 | doi: 10.1021/bk-2017-1257.ch009

organisms under stressed conditions, such as high temperature, drying or freezing. As a result of these well-known phenomena, numerous studies have been carried out in this field to reveal the mechanisms of the stabilization, and to apply this protective effect, e.g. in biological material preservation during processing and storage. Globular proteins, for example, provide many important functional and physicochemical properties to food products, by catalyzing reactions, absorbing to the oil or air surface and stabilizing emulsions and foams, forming gels and more (1–3). The functionality of such proteins is strictly dependent on their structure and stability, which are affected by various parameters, such as temperature, pressure, humidity (during processing and storage), and the presence of co-solutes, like sugars or salts. In food and pharmaceutics production, thermal processes like heating, drying, or freezing play a major technological role, serving various purposes. Heating modifies the structure of proteins and their functional properties in solutions, affecting solubility, viscosity, gelation and surface properties. Adding co-solutes that can be either ionic (4) or non-ionic (5) is an effective way to protect from denaturation, or to solubilize proteins, thereby controlling protein’s functionality. The influence of low-molecular weight solutes on proteins has been studied intensively using different techniques, including NMR (6–9), Raman and IR spectroscopy (6, 10–15), density and sound velocity measurements (16–22), circular dichroism (6), fluorescence spectroscopy (23), light scattering (11), calorimetry (6, 17, 18, 24, 25) and molecular dynamics simulations (22, 26–31). Despite the fact that stabilizing effects of osmolytes are being investigated for about a century, this issue still remains a matter of debate. A general mechanism of protein stabilization by osmolytes in aqueous solutions suggested in literature is known as “The preferential interaction theory”, by Timasheff and co-workers (32–35). This classical view focusses on the relative interactions of the protein with water vs. the protein with the cosolute: co-solutes which are preferentially adsorbed onto the protein, solubilize and denature it, while those which lead to the preferential hydration of the protein, tend to protect the globular native state of proteins in aqueous solutions. On the basis of extensive studies Timasheff and his colleagues had conducted for more than half a century, he postulated that kosmotropic osmolytes are preferentially excluded from the protein surface, whereas water molecules accumulate near its surface. Thus, sugars (known noionic kosmotropes) lead to preferential hydration of the protein, i.e., at equilibrium, there is a higher water concentration near the protein compared to the bulk (19, 33, 36). To sustain this sugar concentration gradient, there is a need for energy investment, so the resultant state of the solution is energetically higher (and less favorable), than the state in the absence of the sugar. As a consequence, a preferentially excluded solute drives the equilibrium between the native and denatured states of the protein toward the native state – a state of the protein that has lower surface area exposed to water than the denatured state. This is well demonstrated by earlier works that showed that kosmotropic osmolytes decreased the apparent specific volume and adiabatic compressibility of several native proteins, which proved that they became more compact (33, 37–39). Concordingly, O’Connor et al. (40) measured the change in free energy of RNase and α-lactalbumin in the presence of sugars, Barreca et al. (41) found 172 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the consequently expected changes in the hydrodynamic volume of BSA during heating in the absence and presence of trehalose, and Saunders et al. (41) showed by CD technique that isothermal titration of polyol osmolytes into thermally- or acid-unfolded protein causes it to fold. However, the preferential interaction theory focusses on the relative strength of interactions with the protein by the solvent and the cosolvent, while in some cases understanding the interactions between the solvent and co-solvent is the key to explaining protein stability in that solvent-cosolvent system. Consequently, the theory cannot fully explain, for example, why different sugars (and in particular, different stereoisomers of the sugar molecule) increase protein thermostability to different extents. Earlier works already reported that different carbohydrates differ in their hydration properties in binary aqueous solutions (7, 8, 16, 42, 43) differences that were expressed in terms of nh, isentropic compressibility, partial molar expansibility and partial molar volume. Newer works based on advanced techniques that analyze the system on a molecular or atomic level, also stress the importance of investigating sugar-water interactions for the understanding more complex biological systems (14, 15, 26, 40, 44, 45). Thus, sugar properties in water are important contributors to the preferential hydration of proteins in solutions, playing an important role in the explanation of protein stabilization. Therefore, in comparing sugars stabilizing effect of a protein under study, it may be preferable to compare sugars that differ in only one hydration-related property at a time, in this case- sugar stereochemistry. In our group, Shpigelman et al. (46, 47) have found a strong correlation between the hydration numbers of four isomeric sugars, galactose, glucose mannose and talose in aqueous solutions, and the extent of cloud-point depression of a synthetic polymer, poly-N-isopropylacrylamide (PNIPA), which served as a model for certain attributes of a protein. The main effect of either rising sugar concentration, or rising nh, was a shift of the coil-to-globule transition of PNIPA to lower temperatures. I.e., the more the stereochemical structure of a sugar promotes its higher hydration, the more it would promote the globular state of the polymer in its presence. We expected that a similar correlation would be observed in case of a protein dissolved in sugar solution, i.e. sugar will promote the protein’s globular state, thus retard its unfolding induced by high temperatures, and this stabilization effect will be enhanced with both sugar concentration, and the nh of the sugar. Molecular structures of the three monosaccharides used in the above work are represented in Figure 1.

Figure 1. Isomeric monosaccharides (aldohexopyranoses). From left to right: α - D(+)mannose, glucose, and galactose. 173 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Thus, in the present study, we have chosen to work with β-lactoglobulin (β-lg). Bovine β-lg is a major whey protein that is used as a food ingredient and its secondary and tertiary structures are well defined. According to X-ray crystallography (48), the structure of a β-lg monomer consists of nine anti-parallel β-strands, eight of which wrap around to create a conical barrel or calyx. The 9th strand participates in the formation of a protein dimer (at neutral pH), and finally, there is a 3-turn α-helix on the outer surface of the calyx (48). It’s secondary structure consists of approximately 50% β-sheet, 15% α-helix, 20% turns and 15% random coil (10). The process of thermal denaturation of β-lg is complex and has been studied quite extensively. According to literature (48), this protein appears to denature first through dissociation of the dimer (at neutral pH), then, at higher temperatures unfolding of the alpha-helix commences, which reveals a free thiol that acts as the initiator of a sulfhydryl-disulfide interchange chain reaction, eventually leading to covalent aggregation, in addition to hydrophobic interactions-based aggregation. In this paper we report an experimental study aimed at shedding light on the mechanism of protein thermal stabilization by low molecular-weight sugars. For this purpose, we performed thermodynamic characterization of the possible sugar-β-lg interactions, using Isothermal Titration Calorimetry (ITC). We also determined a quantitative relationship between the hydration numbers of sugar isomers (mono- and di-saccharides) and their effect on thermal stability of proteins, utilizing microcalorimetry (DSC) and Fourier-Transform Infra Red (FTIR) spectroscopy techniques.

Experimental Section Materials Bovine β-lg was obtained from Davisco Foods International (Le Sueur, MN, USA), dialyzed against double de-ionized water and freeze-dried. Sugars were purchased from Fluka and Sigma Aldrich, and stored in a desiccator at room temperature. All solutions were made by weight in double de-ionized water to which 0.02% (w/v) sodium azide was added as a preservative.

Methods Isothermal Titration Calorimetry (ITC) Titration experiments were performed using a VP-ITC instrument (Micro-Cal Inc., Northampton, MA, USA). Mannose, glucose and galactose solutions at initial concentrations of 0.5 molal (0.46 M at 25°C) were injected into the sample cell (approximate volume 1.47 ml) which was filled with 1mM aqueous β-lg solution. As blanks, sugar solutions were injected into the sample cell, which was filled with water only, and also water was injected into the protein solution. These 174 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

two blanks were subtracted from the sample titration curves to eliminate heat of dilution effects, so that only the protein-sugar interactions would be accounted for. In the urea experiment - its concentration was 0.4 M. Several important injection parameters were as following: injection volume 9 μL, number of injections 30, equilibration time between injections 240 sec, stirring speed 307 rpm and cell temperature 25°C.


Differential Scanning Calorimetry (DSC) DSC measurements were performed with VP-DSC microcalorimeter (Micro-Cal Inc., Northampton, MA, USA). Sugar solution concentrations ranged from 0.25 to 1.0 molal for monosaccharides and from 0.05 to 0.2 molal for disaccharides. Concentration differences between mono- and di-saccharides originated from cellobiose limited solubility (relative to other two isomers). β-lg was dissolved at a constant concentration of 2 mg/ml in the sugar solutions and stirred over night at room temperature for complete dissolution. Degassed protein solutions were scanned in the DSC against the respective sugar solutions as references. Solutions were heated from 50°C to 100°C at a heating rate of 1°C/min. The temperature of the transition peak maximum in the thermogram (Td) was assigned to the denaturation temperature of the protein. Raw thermograms were analyzed by Micro-Cal Origin® software.

Fourier Transform Infra Red (FTIR) Spectroscopy To measure the effect of sugars on the conformation of a protein during its thermal treatment, a chemometric method based on a multivariate Partial Least Squares (PLS) analysis was used. This method correlates several spectral parameters with the reference values of a calibration set of spectra, thus providing a higher degree of precision (10, 49, 50). Calibration samples were prepared by mixing known amounts of two mother solutions: a solution of native β-lg at a concentration of 5 mg/ml and a solution of the denatured protein, which was obtained by incubating the native protein solution at 100°C for 5 minutes and then cooling rapidly. β-lg solution was filtered through 0.22 micron Millipore filter and its concentration after filtration was determined by measuring absorbance at 278 nm and using extinction coefficient of 0.96 L*cm1*gr-1. Thus, its final concentration was 4.2 mg/ml. This way, different contents of native and denatured structures were obtained in one sample. The content of native structure in the calibration samples ranged between 0% (only denatured solution, assuming complete denaturation occurred during the heat treatment) and 100% (only native β -lg solution, assuming the protein used was purely native, and had undergone no heat treatment). These assumptions are only approximately correct, but for the purpose of comparing the effects of sugars, only relative values are required, therefore these assumptions were sufficiently valid. The compositions prepared were at intervals of 10%. Calibration measurements were done at 25°C, in duplicates. 175 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Experimental samples comprised solutions of β-lg in different sugars (each at several concentrations), which underwent the same thermal treatment (100°C, 5 min). The FTIR instrument used (Tensor 27, Bruker Optics, MA, USA) was equipped with CaF2 windows sample cell of 7 micrometer path length, and a MCT detector which was cooled with liquid nitrogen. Additionally, the instrument was purged with nitrogen gas during the experiments to eliminate water vapor. For each spectrum, a 60-scans interferogram was collected at a single beam mode and at 4 cm-1 resolution. A first derivative with 9 smoothing points was applied on protein spectra. Measurements were performed at constant and controlled temperature of 25°C.

Results and Discussion The main aim of the project was to determine a quantitative relationship between the increase in thermal stability of a protein and the stereochemistrydependent hydration number (nh) of sugar isomers. We hypothesized that the protective effect of a sugar on a protein increases with sugar hydration, i.e. the larger the nh, the more kosmotropic the sugar, and the stronger its protective effect on the protein’s globular conformation during heating. As a preceding step, we aimed at measuring the thermodynamics of possible binding interactions between the sugars and the protein macromolecule, to determine whether there is any direct interaction occurring between them. Our hypothesis was that there is no significant binding, and the effects of sugars are mainly mediated via their hydration. We used ITC to examine this hypothesis.

ITC Study - Measurement of Interactions between Sugar and β-lg Raw ITC plots of titrations of glucose and urea are presented in Figure 2. Urea was studied for comparison, as a known chaotropic cosolute (in contrast with the kosmotropic behavior of sugars). As can be seen, titrations of glucose solution into the protein solution, Figure 2(a) or into water, Figure 2(b), were exothermic. Titration of water into the protein solution, Figure 2(c), was expectedly negligible compared to other titrations. Urea, on the other hand, gave endothermic peaks when diluted in water, or added into the protein solution, Figure 2(d) and (e), respectively. Raw ITC results of galactose and mannose isomers were similar to those of glucose. It is interesting to compare sugars’ titration results with those of urea, a nonionic protein denaturant. When observing ITC curves of the three isomeric monosaccharides in Figure 3, after blank subtractions, sugars gave slightly endothermic and rather constant heat of interaction with the protein. Urea, in contrast, gave an exothermic heat of interaction, and its titration curve showed a “Langmuir-like” binding curve, rising towards saturation, in line with the known 176 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


fact that urea shows preferential adsorption to proteins. It can be seen that overall there is no measurable attractive interaction between the sugars and the protein and the net enthalpies of this interaction are positive and very close to zero. Moreover, we found no significant differences between the titration curves of the three monosaccharides (experiments were repeated at least twice and also at 1 molal of the sugars). Finally, no binding model could be fit to the titration curves of the sugars shown in Figure 3. The exothermic binding curve of urea to β-lg suggests of weakly attractive interactions, probably by hydrogen bonding. Furthermore, we managed to fit the urea data to a one-type-of-sites binding model with the binding constant in the order of 102 M-1, and enthalpy of -4 cal/mol, Figure3, right. It has to be stressed however, that unlike the ordinary binding with typical binding constants of 103 to 106 M-1, interactions of non-ionic co-solutes with proteins might have to be treated differently. According to Schelmann’s review (51), typical interaction constants in this case are of the order of 0.01 to approximately 1 M-1. For example, average binding constant of urea with T4 lysozyme was reportedly ~1.2 M-1. Conversely, for sugars, interaction constants with the protein are slightly less than unity. Moreover, binding constants greater than 1, indicate a favorable binding, whereas values less than 1 (which are characteristic to kosmotropic osmolytes) indicate preferential hydration of the protein.

Figure 2. Raw ITC plots of sugar and urea titrations into β-lg solution. (a) Titration of glucose into β-lg solution, (b) glucose into water, and (c) water into β-lg; (d) titration of urea into β-lg solution, (e) urea into water.

177 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 3. Interaction curves of β-lg with isomeric monosaccharides or with urea. β-lg was titrated with (●)-urea, (■)-glucose, (○)-galactose, and (×)-mannose.

The interaction of proteins with co-solvent components is a balance between excluded volume (i.e. bulky cosolutes reduce the mixing entropy, hence are worse cosolvents for the protein compared to water) and weak binding (attractive interactions, which lead to a more negative mixing enthalpy with the protein). It is the sum of these two effects that controls the final behavior of a protein in solution; when the dominant effect is the binding, the co-solute is a denaturant. When the dominant effect is the excluded volume, the co-solute is a stabilizing osmolyte (as in case of saccharides). It is important to note that the excluded volume of sugars, due to their more bulky structure, compared to water, is enhanced by the fact they are relatively strongly hydrated (22), hence behave as if they are in fact even more bulky, leading to a more pronounced, and stereochemistry-dependent, excluded volume effect. To rule out the possibility, that the lack of binding observed at 25°C was coincidental, and that entropically-driven binding is actually possible at other temperatures, we performed ITC measurements of β-lg with glucose or urea 178 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


as a titrant at three different temperatures. By carrying out ITC experiments at different temperatures, it is also possible to shed more light on the type of interactions between the protein and the osmolyte (52, 53). Figure 4 shows titration curves for glucose at 3 different temperatures. These curves do not show any significant dependence on temperature, and no binding model could be fit at any of the temperatures. More pronounced differences were observed between the interaction curves of urea at different temperatures, though these differences were quite small.

Figure 4. titration curves of β-lg with glucose or urea at different temperatures. Titrations with glucose are marked by solid markers, and titrations with urea – by open markers. Titration temperatures were (■,□) 25°C, (●,○) 30°C, (▲,4) 35°C 179 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Consequently, our results, which show no binding of the sugars to the protein, support our hypothesis that the stabilizing effect of sugars must be exerted indirectly, via the water. We hypothesize that the more hydrated a sugar isomer is, the larger its excluded volume effect is expected to be, hence the worse a co-solvent it would be for the protein.


DSC Study - The Effect of Sugars on the Denaturation Temperature of β-lg

To investigate the influence of sugar stereochemistry on the protection against β-lg denaturation we used DSC technique in which the denaturation temperature, Td, of a protein in various solutions can be accurately determined. There are many reports of β-lg thermal denaturation in the presence of different solutes. For example, Matheus et al. (13) reported denaturation temperature of 72.0°C of β-lg (5 mg/ml, in a pH 7.2 phosphate buffered saline) as measured by DSC at a heating rate of 1°C per minute. Burova et al. (24) described a transition temperature of 80°C of 2.24 mg β-lg/ml (dissolved in a pH 6.6 phosphate buffer) and Boye et al. (54) found that increasing glucose concentration from 100 to 500 g/l caused an increase of 6.7°C in denaturation temperature of β-lg (i.e. from 78.3 to 85.0°C). It is worth noting that according to literature, the denaturation temperature of β-lg is independent of protein concentration in the range measured (0.1 – 3 mg/ml) (24). The concentration of protein used in our DSC measurements, 2 mg/ml, was within this range. The denaturation of a protein exposes a large surface area and reveals apolar groups to the solvent, as a result of unfolding. This exposure of peptide groups may change the interaction of a protein with the solvent. Cosolutes which have more favorable interactions with the protein at its unfolded state may induce its unfolding, and vice versa (5). While observing the change in specific heat, using a differential scanning calorimeter (DSC), as a protein solution is heated, the endothermic peak observed due to this exposure of less polar groups indicates its denaturation. The temperature of the maximum point in this curve has been referred to here as the denaturation temperature (Td). We carried out DSC measurements β-lg in pure water, and in rising concentrations of the three aldohexoses under study. The difference between Td in a sugar solution and that in water was defined ΔTd. The results are presented in Figure 5. Figure 5 shows a close-to-linear dependence of ΔTd on sugar concentration. The slope of the linear fit was referred to as Kd, and it describes the strength of thermal stabilization conferred to a protein by the sugar. The inset of Figure 5 shows thermograms of aqueous solutions of β-lg in the presence of increasing concentrations of the monosaccharide galactose. It can be seen, that increasing galactose concentration shifts the endothermic peak of a protein denaturation to higher temperatures. Thus, transition temperature of β-lg increased from 82.9°C in the absence of sugar to 87.2°C in the presence of 0.5 molal galactose and to 89.7°C in the presence of 1 molal of the sugar. 180 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 5. ΔTd vs. monosaccharide concentration ,(■)-galactose, (♦)-glucose, (▲)-mannose. The slope of the linear fit, termed Kd, describes the strength of thermal stabilization conferred to a protein by the sugar. Inset: DSC denaturation thermograms of β-lg in the absence, and in the presence of rising galactose concentrations. Td values are marked above the peaks.

When comparing the three sugar isomers, in terms of Kd, we see that galactose was most effective in protecting the protein against thermal denaturation, followed by glucose, and mannose was least protective. We hypothesized (22) that the protein-solvent-quality of each sugar stereoisomer is related to the hydration of the sugar, and may be quantified in terms of the sugar’s nh. Fig 6 plots Kd as a function of aldohexose nh. This correlation is in the same order, but in the opposite direction compared to correlation Shpigelman et al. (47, 55, 56) found with PNIPA and these three isomeric aldohexoses, and also with talose (47). In these studies we found that the higher the nh, the more negative the slope (Km). Similarly, in another study from our group (Manukovsky et al. (57)), we found the same order of effect of the three isomers studied here, on the deswelling of PNIPA gels, and on sugar partition between the inside and outside of the gel. 181 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 6. Kd as a function of aldohexose hydration number (nh). Results are based on triplicates, error bars represent standard error.

The proposed explanation for the opposite trend in case of a protein compared to PNIPA is as follows: PNIPA presents LCST behavior, i.e. it goes through a coilto-globule transition upon heating in aqueous solution, so the soluting-out effect of the sugars causes a downward shift of this phase transition temperature. However, when a globular protein is heated, it behaves according to a UCST pattern, and goes from a “globular” to an “unfolded” conformation. In this case, the effect of the sugar is reversed, as the fact it is a worse cosolvent for the protein, means that it promotes the globular over the coil conformation, i.e. it shifts the transition to higher temperatures. Therefore, in both cases, the more hydrated the sugar, the worse a co-solvent it is for the polymer, and the more it promotes the globular conformation of the polymer. In the case of the globular protein, this means better protection of the protein from thermal denaturation/unfolding. We have recently proposed the “templating” effect (22) to explain the effects of different sugar stereoisomers on water structure and on polymers in the ternary solution. We have shown that sugars behave as nonionic kosmotropes thanks to the fact that they form stronger hydrogen bonds with water compared to waterwater bonds (22). We have shown that the better a sugar isomer fits into the ideal tetrahedral structure of water, as embodied in hexagonal ice, the better a template it forms for cooperative hydration, hence the higher its nh in binary (sugar-water) solutions, and the better protection it provides to the protein in a ternary solution (22). 182 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 7. Kd values vs. the average number of equatorial OH groups of the isomeric sugars.

Furthermore, the different influence of sugar isomers can be explained in terms of mixing entropy, or in terms of the "excluded volume effect" mentioned above. The hydrated sugar resembles larger solvent molecules, and from entropic considerations, according to the lattice model, larger solvent molecules have lower mixing entropy with the polymer, and they take up a larger volume from which the polymer is excluded. Consequently, they form worse co-solvents for the polymer, causing it to compact, thereby maximizing intra-polymer interactions, and minimizing polymer solvent interactions, thereby stabilizing the native globular conformation. The excluded volume effect may also explain the increasing “soluting out” effect with increasing sugar concentration, and with rising sugar molecular weight. An important enthalpic effect originates from the fact that in both proteins and saccharides the most common functional groups (amides in proteins, and hydroxyls in saccharides) prefer accepting hydrogens in a hydrogen bond, than donating ones. Water, on the contrary, is equally able to donate and to accept hydrogens in H-bonds. Therefore, a sugar is a worse cosolvent for the protein than water, as instead of offering hydrogens to the protein, it competes with the protein on the available water hydrogens. Another explanation suggested in the literature for the effects of different sugars (including sugar isomers) on water structure and on protein stability, is 183 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the number of equatorial hydroxyls (6–8, 58) in the sugar molecules, as they are considered more solvent accessible. However, it is unlikely that a single structural property of a sugar would dictate its effect on water-structure or its protein stabilizing effects. Herein, we have observed no correlation between the Kd values and the average number of equatorial hydroxyls of the isomeric sugars, Figure 7. In contrast, the hydration number, nh, is a comprehensive consequence of many structural aspects of the sugars. In a similar manner, we studied the isomeric disaccharides maltose, cellobiose and trehalose, which are all di-glucoses, only differing in the glucoside bond position and configuration (maltose- α1-4; cellobiose β1-4, and trehalose α1-1a). As in the case of the monosaccharides, there were also positive ΔTm values in the presence of each of the three disaccharides (an example is given for cellobiose isomer in Figure 8). In this case, there also seemed to be a positive correlation between Kd and nh, (Figure 9), though not all differences were statistically significant, apparently due to the large stereosimilarity, as they are all glucose dimers. Secondly, the molal concentrations of the disaccharides used were much smaller than those of the monosaccharides (due to the low solubility of cellobiose). The lower concentrations used have probably also decreased the significance of the differences, keeping in mind that the stabilizing effect of sugars is mostly manifested at high concentrations (1).

Figure 8. ΔTd as a function of concentration of the disaccharide cellobiose.



Figure 9 shows that trehalose was found here to be a significantly stronger stabilizer of β-lg than maltose. Cellobiose showed a slightly (though not significantly) lower protective effect than trehalose. The lowest hydration number observed by maltose apparently results from its α1-4 “folded” configuration which is responsible for the high prevalence of an intramolecular O2-O3′ hydrogen bond (59), resulting in fewer H-bonds with water, hence lower hydration number.

Figure 9. Kd as a function of disaccharide hydration number. Results are based on triplicates, error bars represent standard error.

FTIR Study - The Effect of Sugars on the Native Structure of β-lg after Heating To elucidate the influence of sugars on β-lg conformation during thermal treatment, FTIR spectroscopy, combined with chemometric analysis based on partial least squares (PLS) regression, were utilized. Figure 10(a) presents infra red spectra of β-lg, showing amide I and II peaaks, and Figure 10(b) shows its magnified amide I peak. Amide I and II peak positions are at 1636.8 and 1550.1 cm-1, respectively. Similar positions are reported by Wang et al. (10) for β-lg in aqueous solution where amide I was observed at 1628 cm-1 and amide II, at 1558 cm-1. Allain et al. (12) measured amide I of β-lg at 1635 cm-1, (experiments were performed at neutral pH).



Figure 10. Infra red spectra of native and denatured β-lg. (a) amide I and amide II, (b) amide I, (c) first-derivative amide I and amide II.

According to Figure 10(b), it can be seen that there is a small shift in the Amide I peak of the denatured β-lg compared to the native protein. It is important to note that the term “denatured” protein refers to an ensemble of conformational states when thermal transition is complete. In practice, a denatured protein is not necessarily equivalent to the completely unfolded protein, which is an idealized state referring to the polypeptide chain in its fully solvated conformation. We preferred the term “denatured” only to indicate the difference As was described in detail in the experimental section, a calibration curve for the quantitative analysis was based on samples of known native contents. Their spectra are presented in Figure 11(a) and (b). A calibration curve in the inset of Figure 11(c) is based on 20 points and has a correlation coefficient of 0.98. The optimal number of ranks (or the principle components, PCs) was 1 (this number is reasonable for our two-component system composed of native and denatured states). When analyzing plots of R2 and RMSE of a model versus number of PCs (not shown), it was verified that higher number of PCs could only give slightly better results of R2 and RMSE, although the improvement was not significant and so the lower number of PCs was chosen to avoid overfitting.



Figure 11. Infra red spectra of the calibration protein samples, (a) amide I and II, (b) amide I, (c) first derivative amide I, (d) calibration curve for the quantitative evaluation of a protein conformational change. X axis represents the actual values of native protein percent (% of native protein in the mixture of native and denatured), and Y axis represents predicted values by the model, based on the chemometric spectral analysis.

When we plotted the content of native conformation vs. sugar concentration, a linear regression was obtained. In Figure 12, an example of galactose is given, while the other two isomers gave similar results. The slope of the regression line of each graph was defined Kdf and used to indicate the strength of thermal stabilization by the sugar, as measured by FTIR (analogously to Kd obtained by DSC). When Kdf values in the presence of the different sugars were plotted as a function of sugar hydration number (Figure 13), the following results were obtained. This positive trend is in agreement with the respective positive correlation obtained by the DSC, though it can be seen that glucose did not differ significantly in the strength of thermal stabilization from mannose or from galactose, but the two extreme sugars, mannose and galactose, were statistically different. Thus, conformational results also support our hypothesis and indicate that the more hydrated the sugar is, the stronger its effect on protein stabilization of the native conformation. 187 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 12. β-lg native conformation content as a function of sugar concentration, by FTIR. To enable a more detailed investigation of the β-lg structural changes after heat treatment, a second-derivative of the infra red spectra was used. This is a resolution-enhancement techniques that permit a separation of the absorbance band into its components. Thus, absorbance band in the original spectrum appears as negative sub-bands (with minima peaks) in the respective second-derivative spectrum. Second derivative of the native amide I at the region of 1600-1700 cm-1, in Figure 14 (Line marked with empty arrows), resolved main structural elements that according to the literature can be assigned to the following secondary structures; 1656.8 cm-1 for α-helix, 1631.8 cm-1 for antiparallel β-sheet structure and 1686.9 cm-1 for intermolecular hydrogen-bonded β-structure (12, 54). As for the spectrum of β-lg after thermal treatment (Figure 14, line marked with solid black arrows), it can be seen that bands that corresponded to β-structures in the native protein, decreased in intensity. This trend is in line with the one reported by Boye et al. (54) who studied β-lg during heating in a transmission cell and reported the loss of these structures during the process. Also, a band that was attributed to α-helix (at 1656 cm-1) decreased in intensity and its shift to lower wavenumbers may indicate the appearance of unordered segments in the denatured protein. In addition, a new band at approximately 1618 cm-1 is due to intermolecular β-sheets that result from the process of aggregation (12). 188 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 13. Kdf as a function of sugar hydration number.

Figure 14. Second-derivative spectra of β-lg. Empty arrows point to the line representing the native protein, solid black arrows point to the line representing the protein heated in water only, and the other lines (grey, no arrows) – protein heated in the presence of various galactose concentrations. 189 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Interestingly, spectra of β-lg that was heated in the presence of various galactose concentrations – their bands lie approximately between the native and the denatured proteins’ corresponding bands, Figure 14 (grey lines, no arrows). This result shows that sugar hinders the conformational changes in the protein induced by heating. Thus, there are milder changes in the bands attributed to β- and α-structures compared to the corresponding bands in the heated protein spectrum in water only. Thus, by monitoring conformational changes in the protein, we have shown that sugars hinder the process of unfolding and stabilize the native secondary structure and conformation.

Conclusions Based on ITC measurements, we conclude that the effect of carbohydrates on a protein is apparently not through binding to the protein, but indirectly – via the water. The exothermic heat of sugar dilution in water is an indication that the interactions of sugars with water are stronger than water-water bonds, as we have recently found strong evidence for by MD simulations (22), which is the main reason sugars are kosmotropes. We further conclude that the endothermic mixing of sugars with the protein supports our hypothesis and indicates that sugar solution is not a favorable solvent for the protein. We believe, this observation forms the basis for the sugar stabilization of a native form of a protein. We found that the hydration number of a sugar (determined based on ultra-accurate density and sound velocity measurements) can predict its stabilizing strength of a protein against thermal stress in aqueous solutions. This emphasizes the importance of sugar stereochemistry on hydration properties of different sugars in explaining their different kosmotropic effects on globular proteins. The hydration of a nonionic kosmotropic cosolute, like sugar, is an important component of the mechanism of its stabilizing effect on the protein in aqueous systems. Different orientations of hydroxyl groups in a sugar stereoisomer form a better or a worse template, respectively leading to more or less cooperative hydration, and consequently lead to higher or lower hydration number (the number of water molecules in the sugar-water cluster). The more hydrated the sugar is, the worse a cosolvent it is for the protein compared to water, mainly due to the large size of the hydrated sugar (that causes lower mixing entropy with the protein, and to an excluded volume effect, which is even stronger for higher molecular weight saccharides). The larger the hydration number, the worse a cosolvent the sugar is, thus the more it favors the compact globular state of the protein. This leads to shifting the denaturation temperature to higher values. This conclusion is also supported by FTIR protein secondary structure change measurements, i.e., the larger the hydration number, the higher the residual content of the native secondary structure of a protein which was thermally stressed in the presence of the sugar. Thus, when comparing isomeric sugars, the better a template a sugar is for cooperative hydration, the higher the hydration number, and the stronger its protective effect on the protein. 190 Cheng et al.; Stereochemistry and Global Connectivity: The Legacy of Ernest L. Eliel Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Also for different glucose dimers, a correlation was found between their hydration number and their protective effect against protein thermal denaturation.

Acknowledgments


This work was supported by ISF, the Israel Science Foundation Grant No. 1270/05. We thank Prof. Yuval Shoham for the use of his DSC and ITC equipment, Asst. Prof. Avi Shpigelman for his help with the FTIR, and Dr. Irina Portnaya for her help with the DSC and ITC.

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