Sugar-Mediated Stabilization of Protein against Chemical or Thermal

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Sugar-Mediated Stabilization of Protein Against Chemical or Thermal Denaturation Satoshi Ajito, Hiroki Iwase, Shin-ichi Takata, and Mitsuhiro Hirai J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b06572 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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The Journal of Physical Chemistry

Sugar-mediated

Stabilization

of

Protein

against

Chemical

or

Thermal

Denaturation

Satoshi Ajito1, Hiroki Iwase2, Shin-ichi Takata3, and Mitsuhiro Hirai1*. 1

Graduate School of Science and Technology, Gunma University, 4-2 Aramaki,

Maebashi, Gunma 371-8510, Japan. 2

Comprehensive Research Organization for Science and Society, Tokai, 319-1106,

Japan. 3

J-PARC center, Japan Atomic Energy Agency, Tokai, 319-1106, Japan.

* Corresponding Author E-mail: [email protected] (TELEFAX) INT+81 272-20-7551 (PHONE) INT+81 272-20-7554

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The protective action of sugars against the denaturation of myoglobin was clarified by X-ray and neutron scattering methods. Different types of sugars such as disaccharides (trehalose, sucrose) and monosaccharides (glucose, fructose) were used. Experimental data and theoretical simulation based on three different solvation models (preferential solvation model, non-preferential solvation model, and preferential exclusion (hydration) model) indicated that sugar molecules were preferentially or weakly excluded from the protein surface and preserved the native protein hydration shell. This trend was more evident for disaccharides. The preferential exclusion shifted gradually to the neutral solvation at higher sugar concentrations. On the protective actions of the sugars against the guanidinium chloride-mediated denaturation, all sugars, starting from the low concentration of 5 % w/v, showed the protective trend toward the protein native structure, especially for the secondary structure. The thermal structural transition temperature of myoglobin was raised by about 4-5 °C, accompanied by amyloid formation, for all hierarchical structural levels. In particular, the oligomer formation of the amyloid aggregates was more suppressed. The above protective action was sugar-dependent. The present results clearly suggest that sugars intrinsically protect the native structure of proteins against chemical and thermal denaturation through the preservative action of the hydration shell.


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1. Introduction The well-known fact that protein denaturation and enzyme deactivation can be prevented by sugars and/or polyols has been widely applied in the fields of biochemistry and food preservation. To explain such protective actions, specific bindings between proteins and those additives, changes in solvent viscosities, and surface tension and free energy changes upon transfer of proteins into those additive solutions have been considered.1-6 In particular, it was pointed out that the structure of water is an important factor, as sugars have a common property as a water structure-forming factor (kosmotrope).7,

8

Here, we use the terms 'kosmotrope'

according to the original notation that solutes stabilize proteins and membranes, namely that kosmotropes stabilize proteins and hydrophobic aggregates in solution and reduce the solubility of hydrophobes.9 Water is always involved in hydrogen bonding, hydrophobic interaction, and electrostatic interaction, which are the main stabilizing factors of proteins. Therefore, it is reasonably considered that these additives prevent denaturation through suppression of perturbation of the hydration structure of the protein. In spite of many experimental studies of the effects of sugars on protein structures, using various methods such as densitometry, calorimetry, circular dichroism, NMR, etc., there is little direct evidence concerning the interaction between protein and solvent. Therefore, we cannot but say that its mechanism has not been fully elucidated yet. On the other hand, the mechanism by which proteins fold into their native structures is still one of the important problems in biology.10, 11 Stability of proteins in aqueous solutions is affected by the addition of salts and neutral substances and also by the changes in temperature and pressure. However, because it was practically difficult to experimentally observe the structure of a particular protein in concentrated solutions in which large amounts of co-solutes exist, most of the experimental studies of protein unfolding-folding were conducted under dilute-solution conditions. In the present study, using the synchrotron radiation wide-angle X-ray scattering (SR-WAXS) method and the small-angle neutron scattering (SANS) method, we elucidated the effect of sugars on both the hydration (solvation)-shell and structure transition of myoglobin under chemical and thermal denaturation conditions. Myoglobin is one of the well-studied globular proteins in the context of protein-folding,10 because it shows the cross-beta transition accompanying amyloid aggregate formation.12,

13

We had already clarified the initial process of amyloid

formation of apomyoglobin by the SR-WAXS14 and characterized the dynamics of



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apomyoglobin in the helix-to-sheet transition by the combination of SR-WAXS method and the elastic incoherent neutron scattering method.15 Our current results clearly indicate that sugars can stabilize the native protein structure through the protective action of the protein hydration-shell and that the structural transitions both by chemical and thermal denaturation become to proceed cooperatively among the different structural hierarchical levels. The differential protective abilities of different sugars may provide new insights into the biological functions of sugars. 2. Experimental Methods Myoglobin from horse skeletal muscle (Sigma-Aldrich) was used without further purification. The following sugars were used: trehalose (crystalline di-hydrated powder) from HAYASHIBARA Co. Ltd., sucrose, and fructose from Wako Pure Chem. Co. (Osaka, Japan), and D-glucose from Sigma-Aldrich. All other chemicals were of analytical

grade.

The

following

buffers

were

used:

10

mM

HEPES

(N-(2-hydroxymethyl) piperazine-N'-(2-ethane-sulfonic acid)) and 50 mM NaCl (pH 7.0). A myoglobin solution of 8 % w/v was used as the protein stock solution. The protein solution and sugar solutions of different concentrations were mixed in the ratio of 1:3. The final protein concentration as determined by a UV absorption measurement was about 1.75 % w/v. The sugar concentration was varied from 0 % w/v to 43.6 % w/v (to 37% w/v for trehalose). The UV-visible spectrophotometry measurements were carried out using the UV-1800 (Shimadzu Co.). The densitometry measurements were performed using the oscillation densitometry (DMA35, Anton-Paar GmbH) to determine average scattering densities of sugar solutions. SR-WAXS measurements were performed using BL-40B2 spectrometer at the Japan Synchrotron Radiation Research Institute (JASRI, Harima, Japan) and BL-10C spectrometer at the High Energy Accelerator Research Organization (KEK, Tsukuba, Japan). The X-ray wavelength and the sample-to-detector distance were 0.8 Å and 54 cm at BL-40B2, and 1.55 Å and 24 cm at BL-10C, respectively. The X-ray scattering intensity was recorded by the R-AXIS IV detector (30 × 30 cm2 in area, 100 µm in pixel-resolution, from RIGAKU Co.) at BL-40B2, and by the PILATUS3 2M detector （253.7 x 288.8 cm2 in area, 172 µm in pixel-resolution, from Dectris Co.）at BL10C. The exposure time was 10 s at BL-40B2, and 30 s at BL-10C. The temperature of the solutions contained in the sample cells was maintained between 25°C and 85°C using a model mK2000 temperature controller (Instec, Inc., USA). During the measurements, the sample solutions were slowly oscillated to avoid radiation damages.


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The SANS measurements were carried out using the BL15 TAIKAN spectrometer at the pulsed-neutron source of the Materials and Life Science Experimental Facility (MLF) of the Japan Proton Accelerator Research Complex (J-PARC, Tokai, Japan). The neutron wavelength was 0.5 - 6.0 Å at J-PARC. The sample solutions were contained in the quartz cells with 1 mm path length. The exposure time was 30–120 min. Just before the scattering measurements, the sample solutions were filtered to remove aggregates by using a centrifugal filter unit (Merck Co., Germany) with a molecular weight cut-off of 50 kDa. The background correction for WAXS data was executed based on the method as reported previously.16,

17

The distance distribution function, p(r), was obtained by

Fourier transform of the observed scattering intensity, I(q), as

p(r) =

1 2π 2

∞

(1)

∫ rqI(q) sin(rq)dq 0

where q is the scattering vector (q = (4π/λ)sin(θ/2); θ is the scattering angle; λ is the X-ray wavelength. The radius of gyration, Rg, and the zero-angle scattering intensity, I(0), were determined by using the following equation. Rg

2

∫ =

Dmax

0

2∫

p(r)r 2 dr

Dmax

0

I(0) = 4π

∫

Dmax 0

(2)

p(r)dr

p(r)r 2 dr

(3)

where Dmax is the maximum diameter of the particle determined from the p(r) function satisfying the condition p(r) = 0 for r > 0. The Rg and I(0) values were also calculated by using the Guinier plot (q2 vs. ln I(q)). 2. Results and Discussions 2.1. Estimation of excluded molecular volumes and scattering densities of sugars by densitometry In solution scattering methods using X-ray and neutron, the observed scattering function of a solute-particle depends on the difference between the average scattering densities (ASD) of the solute and the solvent, called as contrast (Δρ).18 Therefore, the ASD of the solvent varies depending on the sugar concentration. The ASDs of sugar solvents can be determined from those mass-densities. Figure 1(A) shows the observed mass-densities of sugar solutions depending on the concentrations (% w/w) at 25 °C, using oscillation densitometry. The mass-density ρsugar of the x % w/w sugar solution is given by:



) N "$ xv (100 − x ) vwater &$',. ρ sugar = 1 / + a # sugar + M water $(.+*100 $% M sugar

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(4)

where, vsugar, vwater, Msugar, and Mwater are the volumes and molecular masses of sugar and water molecules, respectively. Na is the Avogadro constant. We determined vsugar by using Equation 4. The specific volume, electron density, and scattering density of sugar molecules are given in Table 1. From Table 1, the ASDs of solvents can be calculated by the following equation.

ASD =

N a ρ sugar " xvsugar ASDsugar (100 − x)vwater ASDwater % $ '' + 100 $# M sugar M water &

(5)

The calculated ASD values of sugar solutions are shown in Figure 1(B). The ASD of myoglobin, 11.9×1010 cm-2 (0.424 eÅ-3) for X-ray, was calculated from the crystal structure of myoglobin (code no. 1WLA registered in PDB19). Further, the contrast, Δρ, can be calculated as shown in Figure 1(C). The values of the ASD and contrast in Figures 1(B) and 1(C) were used for the following scattering function simulation in Section 2.3. 2.2. Protein structure depending on sugar concentration and species The observed wide-angle X-ray scattering (WAXS) curves of myoglobin in sugar solutions are shown in Figure 2, where (A), (B), (C), and (D) are WAXS curves of myoglobin in trehalose, sucrose, glucose, and fructose solutions, respectively. The concentrations of sucrose, glucose, and fructose were varied from 0 to 43.6 % w/v. Due to lower solubility, the trehalose concentration was varied from 0 to 37 % w/v. The WAXS data of (A) and (C) were reproduced from the previous report.20 The observed WAXS curves in the Figure 1 cover the structure of myoglobin at all different hierarchical structure levels. The WAXS curves in the q regions of q < ~0.2 Å-1, ~0.25 Å-1 < q < ~0.5 Å-1, ~0.5 Å-1 < q < ~0.8 Å-1, and ~1.1 Å-1 < q < ~1.9 Å-1 correspond the tertiary structure, inter-domain correlation, the intra-domain structure, and the secondary structure, respectively, including the closely packed side chains.16,

17

In

Figure 2, with increasing sugar concentrations, the scattering intensity in the small-q region below q = ~0.2 Å-1 decreases systematically, which is attributable to the change of the contrast Δρ. However, the Gaussian profile and the slope are mostly maintained, suggesting the preservation of the tertiary structure. On the other hand, the scattering curve in the q region from ~0.25 to ~0.8 Å-1 shows a gradual change due to the change


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of the contrast, which is reasonably explained in the following simulation due to the change of contrast. The profile of the scattering curve in ~1.1 Å-1 < q < ~1.9 Å-1 does not change evidently as this region is less sensitive to the change of contrast. Thus, Figure 2 indicates that the tertiary to secondary structures of myoglobin are preserved well, even at high sugar concentrations. From Figure 2, the radius of gyration, Rg, and the zero-angle scattering intensity, I(0), were estimated. Figure 3 shows square-root of I(0) and Rg as a function of sugar concentrations, where (A), I(0)1/2; (A), Rg. Both I(0) and Rg values were calculated by using Equations 3 and 4, respectively. The I(0)1/2 is known to be proportional to the product of Δρ and V, where V is the volume of a scattering particle18. The linearly decreasing trend of I(0)1/2 with the rise of sugar concentration was clearly observed for all sugars, especially for trehalose. The Rg value is known to be an important index on the protein size and shape. For a globular protein, its value also depends on the density of solvation (hydration) shell surrounding protein surface. For trehalose, sucrose, and glucose solutions, the Rg values initially decreased by about 1.5-2 Å up to the concentration of 20-35 % w/w, followed by a steady increase. For the fructose solution, the increase of Rg started from 15 % w/w. The observed changes are described well by the following simulation. 2.3. WAXS curve simulations based on different solvation models To analyze the effects of sugars on WAXS curves, we carried out WAXS simulation by using the CRYSOL program.21 This program is based on the spherical-harmonics expansion method18 and is known to reproduce experimental X-ray scattering curves of proteins in solutions accurately, as it can take into account the hydration (solvation) shell by using the crystal structure of a protein registered in the PDB.22 In the CRYSOL program, we can vary the ASD of solvent and the contrast of the hydration (solvation) shell with 3 Å in the width. Based on the myoglobin PDB file (1WLA19) and the previous experimental and theoretical studies on the effects of sugars on proteins, we considered three different cases for protein hydration (solvation) in the present WAXS simulation.23 The first case is that hydration-shell water molecules are preferentially replaced by sugar molecules, meaning the preferential arrangement of sugars surrounding the protein surface. Hereinafter, this case is called the preferential solvation model (Model-1). Such an arrangement would significantly raise the contrast of the solvation shell with increasing sugar concentrations. The second case is that sugar molecules are preferentially excluded from the hydration-shell region of the protein



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owing to the hydration repulsion force. In this case, the hydration-shell density (contrast) is preserved and keeps a constant value (called hereinafter as the preferential exclusion model, Model-2). This case is equivalent to the so-called preferential hydration.1,

2, 24

The above two cases correspond extreme cases that would occur

essentially depending on the difference of partition coefficient of water and sugar at the protein surface region. The third case is that within the framework of the preferential hydration, the replacement of hydrated water by sugar is proportional to the volume fraction of sugar multiplied by a certain exchange ratio (less than 1). This case will be hereafter called as the neutral solvation (Model-3). The molecular dynamics simulation study on the effect of glycerol showed that the preferential interaction coefficient for a protein in glycerol/water mixture was essentially linear with the respect to glycerol molality.25 This would be similar to the present study. In the Model-3, sugar molecules are assumed only weakly excluded from the protein surface when the energy of exclusion is no more than the order of thermal energy, kbT. The above three models are presented schematically in Figure 4. Figure 4 depicts the simulated WAXS curves with the rise of the ASD (electron density) of the sugar solvent: (A) preferential solvation model, (B) preferential exclusion model, and (C) neutral solvation model. The ASD of sugar solvent was varied from 9.365×1010 cm-2 to 10.718×1010 cm-2, which corresponded to the sugar concentration from 0 to 43.6 % w/v. The WAXS simulations for different models were executed under the following criteria. For the preferential solvation model in Figure 4(A), the ASD of the solvation-shell was varied from 1.025 to 1.1 times higher than that of the sugar solvent. For the preferential exclusion model (preferential hydration model) in Figure 4(B), the ASD of the hydration-shell was fixed to be from 1.05 to 1.1 times higher than that of bulk water. For the neutral (non-preferential) solvation model, the replacement of hydrated water by sugar is proportional to the volume fraction of sugar multiplied by a certain exchange ratio defined by β. The ASD of the solvation shell was given as follows.

Dshell = Dsugar βV f + Dwater (1− βV f )

(6)

where, Dshell, Dsugar, and Dwater, are the ASDs of the solvation shell, sugar molecule, and hydration shell; Vf, and β are the volume fraction of sugar and the exchange rate β ( ≤ 1), respectively. The β value of 1 means that the replacement of hydrated water by sugar is just proportional to the volume fraction of sugar in the solvent. β = 0 is as same as the case of the preferential exclusion. Figure 4 (C) shows the example of the β value of 0.5


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in the case where the ASD of the hydration-shell was 1.1 times higher than that of bulk water. In Figure 4, the rise of solvent average scattering density (sugar concentration) caused evident changes of the WAXS curves below q = 0.5 Å-1. It should be noted that the WAXS curve for the Model-2 (preferential exclusion of sugar) shows a significant change. In particular, the change of the small-angle scattering intensity (q < 0.1 Å-1) for the Model-2 was much larger than for the Model-1 and the Model-3. Compared with the experimental scattering curve in the range from q = 0.2 Å-1 to q = 0.3 Å-1, the tendency of the change was more similar to that of the simulated WAXS curve in Figure 4 (B) than in Figures 4 (A) and (C). Figure 5 is the comparison between the experimental and theoretical normalized values of I(0)1/2 and Rg obtained from Figures 2 and 4, where (A): I(0)1/2 and (B): Rg. In Figure 5 (A), the changing tendencies of the experimental I(0)1/2 values for all sugar solutions were reproduced quantitatively within the theoretical frameworks of the Model-2 and the Model-3. In particular, the sugar concentration dependence of the I(0)1/2 for the disaccharides agreed well with that of the Model-2 up to the highest concentration. For the monosaccharides, the experimental I(0)1/2 deviated from the preferential exclusion model starting from ~16 % w/v, and above ~28 % w/v, it fell within the range of the neutral solvation model. With increasing sugar concentration (> 25 % w/w), the experimental value approached the theoretical value of the Model-3 at β = 1. It means that sugar molecules are gradually excluded from the protein surface. The experimental Rg values were also mostly described by the Model-2 and Model-3. The shift from the Model-2 to the Model-3 started from ~16 % w/v for fructose, and from ~28-37 % w/v for other sugars. The above simulations indicate the trend that at low sugar concentration sugar molecules are essentially excluded from the protein surface to preserve native hydration-shell, and at high sugar concentration sugar molecules partially penetrate into or replace the hydrated-water surrounding the protein surface. 2.4. Protective action by sugars on chemical (guanidinium-chloride) denaturation of myoglobin It is well known that guanidinium chloride (GdmCl) is one of the strong denaturants widely used in the studies of protein unfolding-folding.9, 26 GdmCl is a chaotrope that increases the strength of hydrophobic interactions by disrupting water structure and that unfolds proteins. It also destabilizes hydrophobic aggregates and increases the solubility of hydrophobes.27 In contrast, sugars behave as kosmotropes that stabilize proteins and hydrophobic aggregates in solution, thereby reducing the solubility of hydrophobes.



Therefore, in the present systems, both factors were expected to compete with each other by either increasing or decreasing the strength of hydrophobic interactions. Figure 6 presents the GdmCl concentration dependence of the WAXS curves of myoglobin in sugar solutions, where (A): sugar free, (B): 5.1 % w/v trehalose, (C): 10.4 % w/v trehalose, (D): 10.4 % w/v, (E): 10.4 % w/v, and (F): 10.4 % w/v. The insets depict the distance distribution function (p(r)) obtained by the Fourier transform of the observed WAXS curves using Equation 1. As shown previously,16, 17 the observed WAXS curve in each q-region reflects the protein structure at a different hierarchal structure level, namely, the tertiary structure (q < ~0.2 Å-1), the internal structure (~0.25 Å-1 < q < ~0.8 Å-1), and the secondary structure (~1.1 Å-1 < q < ~1.9 Å-1), respectively. In the case of a sugar-free protein solution (Figure 6 (A)), upon increasing the GdmCl concentration from 0 to 2.75 M we observed that the scattering profile in the small-scattering-angle region (q < ~0.2 Å-1) and in the medium-scattering-angle region (~0.25 Å-1 < q < ~0.8 Å-1) began to change from a modulated curve to a monotonic curve starting from ~1.25 M. This clearly indicates that GdmCl induces denaturation of both the tertiary and internal structures of myoglobin. However, the collapse of the secondary structure (~1.1 Å-1 < q < ~1.9 Å-1) is relatively small, suggesting that the secondary structure is partly preserved at the highest GdmCl concentration of 2.75 M in the present experiment. The starting concentration of GdmCl which is capable of promoting a change the WAXS curve in the tertiary and internal structure regions was clearly raised by ~0.25 M for 5 % w/w sugar solution and by ~0.5 M for 10 % w/w sugar solution. These data indicate the preservation of the native protein structure by sugars. This trend was more evidently seen in the internal structure region. The p(r) function can reliably reflect the tertiary structure of proteins. Upon increasing GdmCl concentration, the p(r) function changed noticeably from a symmetric bell-shaped profile to an asymmetric profile with a long tail at a certain GdmCl concentration. The maximum diameter of the protein, Dmax, determined from the p(r) function satisfying the condition p(r) = 0 for r > 0, changed from ~50 Å to ~85-90 Å, suggesting the unfolding of the tertiary structure. The starting GdmCl concentration of the unfolding was higher in order of glucose > trehalose > sucrose ≈ fructose. The above trend agreed with the variation in the Rg value as shown below. We were able to characterize the effect of GdmCl on the polypeptide chain under the presence of sugars. Figure 7 presents the Kratky plots (q2I(q) vs. q) of the WAXS curves in Figure 6. The Kratky plots are widely applied to characterize the rigidity of polymer chains.28 In the sugar-free solution (Figure 7 (A)), the bell-shaped peak at q =


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~0.1 Å-1 became flat with increasing GdmCl concentration, indicating that the globular protein structure turns into an expanded form. The rigidity feature of the peptide backbone as a polymer chain appears in the region larger than q = ~0.25 Å-1. When the GdmCl concentration was raised to 2 M, the modulated curve became a simple line with a slope as long as the condition: q = ~0.8 Å-1 was satisfied. This indicates that the peptide backbone changes from a persistent chain with persistent curvature to a simple persistent chain within the real space distance larger than ~8 Å. On the other hand, in the presence of sugars, the characteristic of a persistent chain with persistent curvature was maintained even at the highest GdmCl concentration, evidently in the cases of trehalose and glucose. Figure 8 depicts the Rg values obtained from the WAXS curves. The decreasing tendencies of the Rg values above 2.5 M GdmCl in the samples with the sugar concentrations of 0 and 5 % w/w are artefacts arising due to the lack of the scattering data in the small-angle region. Thus, from Figure 8, we determined the mid-point GdmCl concentration of the unfolding, cm, by a sigmoidal-function fitting using the Rg values below 2 M GdmCl. The cm values are listed in Table 2. The increment of cm was in the range of 0.15 - 0.2 M at 5 % w/w sugar concentration, and in the range of 0.33 - 0.52 M at 10 % w/w sugar concentration. When comparing the by weight concentrations, the increment of cm for glucose was the highest. When comparing the by molar concentrations, the increment of cm for trehalose was the highest. Interestingly, both of them are aldoses. As we observed the WAXS curves covering the whole hierarchical structure of myoglobin from the tertiary structure (q < ~0.2 Å-1), the internal structure (~0.25 Å-1 < q < ~0.8 Å-1), to the secondary structure (~1.2 Å-1 < q < ~1.9 Å-1), we were able to characterize the denaturation of myoglobin by GdmCl at the different hierarchical structure levels. Here, we used the transition-multiplicity analysis (TMA) method17, 29. This method can afford us an empirical value of the molar fraction of the native structure at the different hierarchical structure levels in the denaturation process by using the following equation. + $ (. & &0 qj - I(q, c) & I(q, cU ) &0 I(q, cN ) Δ ij = ∑ - q j − %α ij q j + (1− α ij ) q j )0 & & q=qi I(q, c) & ∑ I(q, cN ) I(q, cU ) &0 ∑ ∑ -, q=qi q=qi ' q=qi *0/

(7)

where, I(q, cN ) , I(q, cU ) , and I(q, c) are the scattering curves at the initial (native), final (unfolded), and intermediate GdmCl concentrations in a defined q-range of qi-qj Å-1, respectively. The scattering curve I(q, c) in the q-range of qi-qj at an intermediate



concentration was fitted by using a linear combination of

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αij I(q, cN ) and

(1− α ij )I(q, cU ) at the initial and final concentrations. The factor αij was determined by minimizing the Δ ij value. The α ij value means the molar fraction of the native structure in the defined hierarchical structure level corresponding to the q-range of qi-qj. Figure 9 presents the GdmCl concentration dependence of the α values corresponding € to the tertiary, internal, and secondary structures of myoglobin, respectively. The cm at each hierarchical structure level, was obtained from the concentration at α = 0.5 in Figure 9. The cm values are listed in Table 2. It should be mentioned that the cm values determined from the radii of gyration in Figure 8 agree well with those determined by the TMA analysis using the tertiary structure region in Figure 9 (A). In the case of the sugar-free sample, the cm value of the tertiary structure was lower by around 0.2 M compared with those of the internal and secondary structures, suggesting that the tertiary-structure transition proceeds in advance during the GdmCl-induced denaturation. In the case of sugar solutions, the cm value at 10 % w/w sugar concentration raised significantly, by about 0.5 - 1 M. Table 2 shows that the protective action on the secondary structure by trehalose and glucose occurred more clearly compared with the cases of sucrose and fructose. Noteworthily, the addition of GdmCl causes the change of the ASD of the solvent as much as the addition of sugars. The excluded volume of GdmCl determined from the density measurement is 116.6±0.6 Å-3 (data not shown). Then, the rise of the GdmCl concentration from 0 to 2.75 M is assumed to increase the ASD of the solvent from 9.365×1010 cm-2 to 9.533×1010 cm-2. However, this increment is rather small compared with that by the addition of sugars shown in Figure 2. Therefore, the alteration in the WAXS curves in Figure 6 mostly reflects the influence caused by the GdmCl addition, and not by the change of the contrast of the protein. To confirm the WAXS results, we carried out neutron scattering measurements using the inverse contrast variation method. The inverse contrast variation method using deuterated materials30 can avoid the artificial effect on the scattering curves caused by the addition of sugar molecules. As we knew the molecular volume of glucose shown in Table 1, the ASDs (average scattering length density) of non-deuterated glucose (h-glucose) and 97%-atom-deuterated glucose (d-glucose) for neutron were calculated to be 2.67 × 10-10 cm-2 and 7.96 × 10-10 cm-2, respectively. Then, the ASD of the mixture of [d-glucose]/[h-glucose] = 0.706/0.294 (M/M) was expected to match with that of D2O (6.404 × 10-10 cm-2). When we used this mixture, the contrast of the protein in D2O solvent was not affected by the addition of glucose, i.e., the contribution of glucose to the scattering curve was undetectable. Figure 10 shows the observed SANS curves


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depending on the GdmCl concentration, where (A): in D2O solvent without glucose, and (B): in D2O solvent with 5 % w/w glucose. Figure 11 depicts the Rg values obtained from Figure 10. It should be mentioned that the Rg values obtained by SANS are different from those obtained by SR-WAXS. It can be explained by the difference in the contrast profile of myoglobin in D2O (for SANS) and H2O (for SR-WAXS). Namely, the contrast profile of myoglobin including its solvation shell takes a positive value outside and a negative value inside in the case of SANS, and vice versa in the case of SR-WAXS, because in the present experimental condition the ASD of the solvation shell is ~1.1 times higher than that of the solvent. With the rise of the GdmCl concentration, the Rg values varied from 14.1±0.2 Å to 26.3±0.8 Å in D2O solvent without glucose, and from 14.2±0.2 Å to 26.3±0.5 Å in D2O solvent with 5 % w/w glucose. This changing trend agrees well with those observed by the WAXS measurements shown in Figure 8. Myoglobin is a porphyrin-containing metalloprotein which has one heme. By monitoring the absorption of the Soret band of heme (at 409 nm), we analyzed the effect of sugar on the structural environment surrounding the heme. Figure 12 presents the absorption spectrum of myoglobin in the GdmCl solutions, where (A): GdmCl only, (B): GdmCl + 20 % w/w trehalose, and (C): GdmCl + 20 % w/w sucrose. Upon increasing the GdmCl concentration, the absorption peak intensity at 409 nm decreased sharply, indicating the dissociation of heme from the heme-pocket due to the denaturation. Figure 13 depicts the change of the absorption peak intensity depending on the GdmCl concentration. By the addition of sugars in 20 % w/w, the dissociation concentration was raised by about 0.5 M. These results further support the WAXS and SANS results. 2.5. Protective action by sugars on thermal denaturation of myoglobin Myoglobin is known to form amyloid structures in thermal and/or acid denaturation processes.12-14 The sugar solutions tested were trehalose, sucrose, glucose, and fructose. Those concentrations were 10 % w/w and 20 % w/w. Figure 14 presents the temperature dependence of the WAXS curves of myoglobin, where (A): in pure solvent, (B): in 21.7 % w/v trehalose solution, and (C): in 21.7 % w/v glucose solution. The arrows in Figure 15 (A) indicate typical features appearing in the initial process of amyloid transition of myoglobin.12, 13, 31 The arrows at q = ~0.08 Å-1, ~0.58 Å-1, and ~1.34 Å-1 correspond to the formation of an amyloid-oligomer, the pleated-beta-sheet stacking, and the helix-to-sheet (cross-beta-sheet) transition, respectively. At the sugar



concentration of 21.7 % w/v, the oligomer formation was commonly suppressed by all sugars (trehalose, sucrose, glucose, and fructose). Even at the sugar concentration of 10.4 % w/v, the oligomer formation was suppressed for the monosaccharides. In addition, in the cases of glucose and fructose, the pleated-beta-sheet stacking was depressed at 21.7 % w/v. Similar to the above section, the TMA method is also applicable to characterize the thermal stability in the different hierarchical structure levels. Equation 7 can be rewritten as Equation 8, where the concentration c is replaced by the temperature T. + $ (. & &0 qj - I(q,T ) & I(q,TU ) &0 I(q,TN ) Δ ij = ∑ - q j − %α ij q j + (1− α ij ) q j (8) )0 & &0 q=qi ∑ I(q,T ) & ∑ I(q,TN ) ∑ I(q,TU ) &0 -, q=qi q=qi ' q=qi */ where I(q,TN ) , I(q,TU ) and I(q,T) are the scattering profiles at the initial, final, and intermediate temperatures in a defined q-range of qi-qj Å-1, respectively. The scattering curve I(q,T) in a defined q-range at an intermediate temperature is fitted by using a € € linear combination € of α ij I(q,TN ) at the initial temperature and (1− α ij )I(q,TU ) at the €

final temperature. The selected q-ranges were 0.05 – 0.2 Å-1, 0.25 – 0.8 Å-1, 0.55 – 0.65 Å-1, and 1.30 – 1.38 Å-1. These regions correspond to the tertiary structure, the internal € structure, the pleated-beta-sheet stacking, and the helix-to-sheet transition, respectively. The last two regions are specific for the amyloid transition. The mid-point temperature of the thermal transition, Tm, was determined by the temperature at α = 0.5 in Figure 15. The obtained Tm values are summarized in Table 3. In the case of the pure aqueous solvent, the Tm values at the different hierarchal structural levels were within ~78 ± 1 °C, suggesting that the structural transition proceeds cooperatively among all different hierarchal structural levels. On the other hand, with increasing sugar concentration from 0 to 21.7 % w/v, the Tm values for all hierarchal structural levels raised by ~ 4-5 °C. The rise of the Tm value for the pleated-beta-sheet stacking and the helix-to-sheet transition was slightly larger than that for the tertiary and internal structures. Interestingly, the protective action by sugars against thermal denaturation occurs over all the hierarchical structure levels, which contrasts with the case of GdmCl denaturation. 3. Conclusions By X-ray and neutron scattering techniques, and theoretical simulation, we clarified the molecular mechanism of the protective action of sugars against thermal and chemical denaturation of myoglobin. We mainly analyzed the effect of sugar on hydration


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(solvation) of the protein, and the following results were obtained1) Effect of sugars on protein solvation-shell The sugar molecules were preferentially excluded and/or weakly excluded from the protein surface to preserve a native hydration shell. The theoretical simulation based on the preferential exclusion and neutral solvation models qualitatively explained the experimental results. This trend was more evident for disaccharides (trehalose (aldose-aldose) and sucrose (aldose-ketose)), when the concentration was given in % w/v. Upon increasing the sugar concentration above 21.7-27.6 % w/v, the shift from the preferential exclusion (preferential hydration) to the neutral solvation (partial penetration of sugar into the hydration shell region) was observed. In the case of fructose (mono-ketose-saccharide), this shift started from a lower concentration (below ~16 % w/v) compared with other sugars. 2) Protective action by sugar against chemical denaturation of myoglobin During the competitive action of sugars and GdmCl as kosmotrope and chaotrope, the presence of sugars significantly suppressed the protein denaturation, especially for the secondary structure compared with the tertiary and internal structures, except for fructose. The transition concentration increased by ~0.5 – 1 M at 10.4 % w/v sugar. This phenomenon was observed for every sugar. This protective action of sugars was observed even at the low sugar-concentration of 5.1 % w/v. 3) Protective action of sugars against thermal denaturation of myoglobin The thermal denaturation of myoglobin was known to accompany the amyloid transition. In the sugar-free solvent, the destabilization of the internal structure progressed in advance. By the presence of sugars, the transition temperatures at all hierarchical structure levels were elevated to take the similar values, and the transition proceeded cooperatively at all the hierarchal structure levels. Thus, the present results suggest that the protective action of sugars on the protein native structure against chemical and thermal denaturations strongly relates to the preservation of the native hydration-shell. This trend was more evidently seen for trehalose and glucose. From a biological points of view, it is well known that organisms having tolerances against extreme environment produce stress accumulate proteins and/or sugars in cells under the external stress such as high or low temperature, drying, osmotic pressure, and so on. In particular, trehalose has been drawing attention by the relation with biostasis (cryptobiosis, anhydrobiosis), that is the ability of an organism to tolerate environmental changes without having to actively adapt to them.32-35 Trehalose is an alpha-linked disaccharide formed by an α,α-1,1-glucoside bond between two



α-glucose units. As reducing groups are bonded to each other, trehalose does not have reducing properties. The action of trehalose to afford an organism to tolerate environmental changes has been thought to result from the restriction of the intra-and/or-inter-molecular movement by vitrification or from the replacement of water molecules by trehalose.34 In this study, we treated monosaccharide (glucose, fructose) and disaccharide (sucrose, trehalose). From the general knowledge of sugars, the former is reducible and has an ability of the reaction of glycation that results in the covalent bonding of a sugar molecule to protein or lipid molecule without the control of enzyme and impairs or destroys the functioning of biomolecules.36 The later is classified in non-reducible sugar, however, trehalose has a higher glass transition temperature37 and molecular flexibility38 than sucrose. Although the present results show that the protective action of trehalose against protein denaturation has minor difference compared with other sugars, such an intrinsic difference in the physicochemical properties of sugars may be one of the reasons why some organisms favor the accumulation of trehalose against extreme environments.


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AUTHOR INFORMATION Corresponding Author (M.H.) *E-mail: [email protected] (TELEFAX) INT+81 272-20-7551 (PHONE) INT+81 272-20-7554 Notes

The authors declare no competing financial interest. ACKNOWLEDGMENTS The X-ray scattering experiments were performed under the approval of the program advisory committee of the Japan Synchrotron Radiation Research Institute (Proposal No. 2016B1381 & 2017B1435) and under the approval of the PF program advisory committee (Proposal No. 2016G560 & 2017G698). The neutron scattering experiment was done under the approval of the Neutron Scattering Program Advisory Committee (Proposal No. 2017B0218 & 2018A0115). This research project was partly supported by the Grants-in-Aid for Scientific Research of JSPS (the Japan Society of the Promotion of Science) (Proposal No. 16K13722).



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(16) Hirai, M.; Iwase, H.; Hayakawa, T.; Miura, K.; Inoue, K. Structural hierarchy of several proteins observed by wide-angle solution scattering. J. Synchrotron Rad. 2002. 9, 202-205. (17) Hirai, M.; Koizumi, M.; Hayakawa, T.; Takahashi, H.; Abe, S.; Hirai, H.; Miura, K.; Inoue, K. Hierarchical map of protein unfolding and refolding at thermal equilibrium revealed by wide-angle X-ray scattering. Biochemistry 2004, 43, 9036-9049. (18) Stuhrmann, H. B. 1978. Miller, A. Small-angle scattering of biological structures. J. Appl. Cryst. 1978, 11, 325-345. (19) Maurus, R.; Overall, C. M.; Bogumil, R.; You, Y.; Mauk, A. G.; Smith, M.; Brayer, G. D. A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket, Biochem. Biophys. Acta 1997, 134, 1-13. (20) Ajito, S.; Hirai, M.; Iwase, H.; Shimizu, N.; Igarashi, N.; Ohta, N. Protective action of trehalose and glucose on protein hydration shell clarified by using X-ray and neutron scattering. Physica B 2018, doi.org/10.1016/j.physb.2018.03.040. (21) Svergun, D. I.; Barberato, C.; Koch, M. H. J. CRYSOL-a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates, J. Appl. Cryst. 1995, 28, 768-773. (22) Svergun, D. I.; Richard, S.; Koch, M. H. J.; Sayers, S.; Kuprin, S.; Zaccai, G. Protein hydration in solution: Experimental observation by X-ray and neutron scattering. Proc. Natl. Acad. Sci. USA 1998, 95, 2267-2272. (23) Auton, M.; Bolen, D. W.; Rosgen, J. Structural thermodynamics of protein preferential solvation. Proteins: Struct. Funct., Bioinf. 2008, 73, 802-813. (24) Ronsina, O.; Caroli, C.; Baumberger, T. 2017. Preferential hydration fully controls the renaturation dynamics of collagen in water-glycerol solvents. Eur. Phys. J. E 2017, 40, 55. (25) Vagenende, V.; Yap, M. G. S.; Trout, B. L. Mechanisms of protein stabilization and prevention of protein aggregation by glycerol. Biochemistry 2009, 48, 11084– 11096. (26) Bai, Y.; Sosnick, T. R.; Mayne, L.; Englander, S. W. Protein folding intermediates: native-state hydrogen exchange. Science 1995, 269, 192-197. (27) O’Brien, E. P.; Dima, R. I.; Brooks, B.; Thirumalai, D. Interactions between hydrophobic and ionic solutes in aqueous guanidinium chloride and urea solutions: Lessons for protein denaturation mechanism. J. Am. Chem. Soc. 2007, 129, 7346-7353. (28) Kriste, R. G.; Oberthur, R. C. Synthetic polymers in solution. in Small angle X-ray



scattering (Glatter, G. and Kratky, O. eds.). Academic Press, 1982, pp. 387-431. (29) Hirai, M.; Arai, S.; Iwase, H. Complementary analysis of thermal transition multiplicity of hen egg-white lysozyme at low pH using X-ray scattering and scanning calorimetry. J. Phys. Chem. B 1999, 103, 549-556. (30) Knoll, W.; Schmidt, G.; Ibel, K. The inverse contrast variation in small-angle neutron-scattering. J. Appl. Cryst. 1985, 18, 65-70. (31) Malinchik, S. B.; Inouye, H.; Szumowski, K. E.; Kirschner, D. A. Structural analysis of Alzheimer's beta(1-40) amyloid: Protofilament assembly of tubular fibrils. Biophys. J. 1998, 74, 537-545. (32) Elbein, A. D.; Pan, Y. T.; Pastuszak, I.; Carroll, D. New insights on trehalose: a multifunctional molecule. Glycobiology 2003, 13, 17R-27R. (33) Wełnicz, W.; Grohme, M. A.; Kaczmarek, Ł.; Schill, R. O.; Frohme, M. Anhydrobiosis in tardigrades—The last decade. J. Insect Physiology 2011, 57, 577–583. (34) Feofilova, E. P.; Usov, A. I.; Mysyakina, I. S.; Kochkina, G. A. Trehalose: chemical structure, biological functions, and practical application. MICROBIOLOGY 2014, 83, 271–283. (35) Møbjerg, N.; Halberg, K. A.; Jørgensen, A.; Persson, D.; Bjørn, M.; Ramløv, H.; Kristensen, R. M. Trehalose metabolism in plants. Plant J. 2014, 79, 544-67. (36) Bunn, H. F.; Higgins, P. J. Reaction of monosaccharides with proteins: possible evolutionary significance. Science 1981, 213, 222–224. (37) Crowe, J. H.; Carpenter, J. H.; Crowe, L. M. The role of vitrification in anhydrobiosis. Annu. Rev. Physiol. 1998. 60, 73–103. (38) Simperler, A.; Kornherr, A.: Chopra, R.; Bonnet, P. A.; Jones, W.; Motherwell, W. D. S.; Zifferer, G. Glass transition temperature of glucose, sucrose, and trehalose-An experimental and in silico study. J. Phys. Chem. B 2006, 110, 19678-19684.


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Table 1. Excluded volumes, electron densities, and X-ray scattering densities of sugars determined by densitometry. Table 1. Excluded volumes, electron densities, and X-ray scattering densities of sugars trehalose sucrose glucose fructose 3 excluded volume (Å ) 353.3±0.4 355.0±0.2 189.7±0.7 187.0±0.3 3 electron density (e/Å ) 0.5152 0.5127 0.5061 0.5134 10 -2 scattering density (x10 cm ) 14.48 14.41 14.22 14.43

Table 2. Table 2. Mid-point GdmCl concentration of myoglobin unfolding A B C tertiary structure internal structure secondary structure q=0.05–0.20 Å-1 q=0.25–0.8 Å-1 q=1.3–1.8 Å-1 (by fitting) sugar-free 1.38±0.01 M 1.58±0.03 M 1.49±0.07 M 5.1% trehalose 1.55±0.01 M 1.78±0.03 M 1.89±0.06 M 5.1% sucrose 1.53±0.01 M 1.77±0.02 M 1.79±0.08 M 5.1% glucose 1.59±0.01 M 1.79±0.03 M 2.13±0.05 M 10.4% trehalose 1.93±0.01 M 2.00±0.05 M 2.26±0.06 M 10.4% sucrose 1.81±0.01 M 1.89±0.02 M 2.1±0.1 M 10.4% glucose 1.94±0.01 M 2.01±0.01 M 2.49±0.03 M 10.4% fructose 1.76±0.01 M 2.01±0.03 M 1.8±0.1 M

D radius of gyration 1.43 ± 0.01 M 1.61 ± 0.01 M 1.58 ± 0.01 M 1.64 ± 0.01 M 1.93 ± 0.01 M 1.80 ± 0.01 M 1.95 ± 0.01 M 1.76 ± 0.01 M

The transition mid-point concentrations, cm, in the columns from A to C, corresponding to the different hierarchical structure levels of the protein, were determined by the TMA analysis in Figure 9. The cm value in the column D was obtained from the Rg values in Figure 8. The concentration of sugar was given in % w/v.



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Table 3. Table 3. Mid-point temperature of thermal denaturation of myoglobin A B C tertiary structure internal structure sheet stacking q=0.05–0.20 Å-1 q=0.25–0.8 Å-1 q=0.55–0.65 Å-1 (by fitting) sugar-free 78.9±0.1 ˚C 77.3±0.2 ˚C 77.2±0.2 ˚C 10.4% trehalose 82.3±0.2 ˚C 82.3±0.3 ˚C 81.8±0.5 ˚C 10.4% sucrose 81.8±0.3 ˚C 81.4±0.4 ˚C 79.8±0.7 ˚C 10.4% glucose 82.7±0.4 ˚C 83.1±0.7 ˚C 83.8±0.5 ˚C 10.4% fructose 80.6±0.4 ˚C 81.3±0.3 ˚C 80.2±0.9 ˚C 21.7% trehalose 82.8±0.1 ˚C 82.6±0.2 ˚C 82.5±0.2 ˚C 21.7% sucrose 81.7±0.2 ˚C 82.7±0.1 ˚C 81.1±0.3 ˚C 21.7% glucose 83.0±0.2 ˚C 82.9±0.7 ˚C 82.7±0.2 ˚C 21.7% fructose 82.9±0.2 ˚C 83.0±0.7 ˚C 83.1±0.5 ˚C

D cross-β structure q=1.30–1.38 Å-1 76.7±0.5 ˚C 82.5±0.4 ˚C 81.7±0.3 ˚C 82.4±0.4 ˚C 82.8±0.9 ˚C 82.4±0.7 ˚C 82.5±0.4 ˚C 83.1±0.8 ˚C 82.7±0.9 ˚C

The columns from A to D show the thermal structural transition mid-point temperatures, Tm, at the different hierarchical structure levels of the protein. These values were determined by the TMA analysis in Figure 15. The columns C and D correspond to the q-ranges for typical features of amyloid transition, namely, pleated sheet stacking, and cross-β structure, respectively. The concentration of sugar was given in % w/v.


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[Figure captions] Figure 1. Mass density and average X-ray scattering density (ASD) of the sugar solutions at 25 °C, and contrast (Δρ) of myoglobin. (A) Mass density (g/mL); (B) Average scattering density (cm-2); (C) Contrast (cm-2). Sugars are trehalose, sucrose, glucose, and fructose. The lines in (A) depict least-square-fitting for the estimation of excluded volumes of sugar molecules. Figure 2. Wide-angle X-ray scattering (WAXS) curves of myoglobin depending on the sugar concentration (% w/v). (A), (B), (C), and (D) are WAXS curves of myoglobin in trehalose, sucrose, glucose, and fructose solutions, respectively. The protein concentration is 1.75 % w/v in 10 mM HEPES buffer at pH 7 at 25 °C. The sugar concentration was varied up to 43.6 % w/v for sucrose, glucose, and fructose, and up to 37 % w/v for trehalose. Figure 3. Zero-angle scattering intensity (I(0)1/2) and radius of gyration (Rg) depending on the sugar concentrations. (A), I(0)1/2; (B), Rg. Figure 4. Simulation of WAXS curve depending on the average scattering density that corresponds to the rise of the sugar concentration from 0 to 43.6 % w/v, where (A), (B), and (C) correspond to the different types of the solvation models. (A) Preferential solvation model (Model-1, preferential replacement of protein hydration-shell water by sugar molecules); (B) Preferential exclusion model (Model-2, preferentially exclusion of sugar molecules from the protein hydration-shell region, preferential hydration). (C) Neutral (non-preferential) solvation model (Model-3, replacement of hydrated water by sugar is proportional to the volume fraction of sugar). Each model is shown schematically in each figure. The WAXS simulations were performed by using the CRYSOL program.



Figure 5. Comparison between the normalized values of the experimental and theoretical I(0)1/2, and Rg. (A) I(0)1/2; (B) Rg. The frames depict the theoretical values in the different solvation models; Model-1 (preferential solvation), Model-2 (preferential exclusion), and Model-3 (non-preferential solvation), respectively. Figure 6. Variation of WAXS curve of myoglobin in sugar solutions by guanidinium chloride (GdmCl) denaturation at 25 °C. GdmCl concentration was changed from 0 to 2.75 M. (A) Sugar free; (B) 5.1 % w/v trehalose; (C) 10.4 % w/v trehalose; (D) 10.4 % w/v sucrose; (E) 10.4 % w/v glucose; (F) 10.4 % w/v fructose. The insets show the distance distribution functions, p(r), obtained by the Fourier transform of the observed WAXS curves. The p(r) function reflects well a tertiary structural change. Figure 7. Variation of the Kratky plot of the WAXS curve depending on the GdmCl concentration. (A), (B), (C), (D), (E), and (F) are as in Figure 6.

Figure 8. Change of the radius of gyration, Rg, of myoglobin in sugar solutions in the GdmCl denaturation process. The Rg values were evaluated from the WAXS curves in Figure 6 and others. The lines are fitting curves of a sigmoidal function. Figure 9. Molar fraction α (α ≤ 1) of the native structure (at 25 °C) of myoglobin at an intermediate GdmCl concentration in the denaturation process as determined from the scattering curves in different q-ranges by using the TMA analysis. The selected q ranges of (A) (0.05 - 0.2 Å-1), (B) (0.25 - 0.8 Å-1), and (C) (1.3 – 1.8 Å-1) correspond to the tertiary structure, the internal structure, and the secondary structure, respectively. The transition mid-point concentration of the unfolding, cm, at each hierarchical structure level, was obtained from the concentration at α = 0.5. Figure 10. Neutron scattering (SANS) curve of myoglobin depending on the GdmCl concentration


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at 25 °C. (A) In D2O solvent; (B) In D2O solvent with 5.1 % w/v glucose. By using the mixture of [deuterated glucose] / [non-deuterated glucose] = 0.706/0.294 (M/M), the contribution from glucose on the SANS curve was matched out and invisible. Figure 11. Change of the radius of gyration, Rg, of myoglobin in sugar solutions in the GdmCl denaturation process observed by SANS. Figure 12. Absorption spectrum of myoglobin in the GdmCl solutions, where (A) GdmCl only; (B) GdmCl + 21.7 % w/v trehalose; (C) GdmCl + 21.7 % w/v sucrose. Figure 13. Change of the absorption peak intensity of the Soret band of heme at 409 nm depending on the GdmCl concentration. The peak intensity was normalized by the absorption peak at 280 nm. Figure 14. Temperature dependence of the WAXS curve of myoglobin. (A) In water solvent, (B) In 21.7 % w/v trehalose, (C) In 21.7 % w/v glucose. The insets enlarge the WAXS curves in the secondary structure region. The arrows indicate the typical features in the scattering curve appearing at the initial process of amyloid transition, namely, the pleated-beta-sheet stacking and the helix-to-sheet (cross-beta-sheet) transition. Figure 15. Molar fraction α (α ≤ 1) of the native structure (at 25 °C) of myoglobin at an intermediate temperature in the heating process at different hierarchal structure levels as determined by using the TMA analysis. The selected q ranges of (A) (0.05 - 0.2 Å-1), (B) (0.25 - 0.8 Å-1), (C) (0.55 – 0.65 Å-1), and (D) (1.3 – 1.38 Å-1) correspond to the tertiary structure, the internal structure, the pleated-beta-sheet stacking, and the helix-to-sheet transition, respectively.



Figure 1

mass density (g/ml)

1.20

(A)

1.15 1.10 trehalose sucrose glucose fructose

1.05 1.00

11.5 -2

11.0

10

(x 10 cm )

average scat. dens.

12.0

10.5

myoglobin

(B)

10.0 9.5

-2

2.0

10

1.5

(x 10 cm )

contrast

2.5

(C)

1.0 0.5 0

10

20

30

40

50

sugar conc. (% w/w)

logI(q) (arb. units)

Figure 2

(B)

(A)

sugar conc.(% w/w) 0

1

5

1

10 15 17.5 20


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

22.5

(C)

(D)

25 30 32.5 35 37.5

1

1

0.1

q (Å-1)

1

0.1

q (Å-1)

1


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Figure 3

I(0)

1/2

(-)

1.8

1.4

(A) 1.0 trehalose sucrose glucose fructose

0.6

0.2 22.0

Rg (Å)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(B)

20.0 18.0 16.0 14.0 0

10

20

30

40

sugar conc. (% w/v)



Figure 4


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Figure 5 1.0

normalized I(0)1/2 (-)

0.9 0.8

(A)

0.7 0.6

trehalsoe sucrose glucose fructose

0.5 0.4

Model-1

Model-3

0.3 Model-2

0.2 1.4 1.3

normalized Rg (-)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(B)

1.2 1.1

Model-1

1.0 1.0

Model-3

0.9 0.8

Model-2 0

10

20

30

40

sugar conc. (% w/v)



(C)

p(r) (-)

(B)

p(r) (-)

(A)

p(r) (-)


Figure 6

GdmCl conc. (M) 0

0

0

20 40 60 80 r (Å)

0

20 40 60 80 r (Å)

20 40 60 80 r (Å)

0.25 0.50

1

1

1

0.75 1.0

(F)

1.50

p(r) (-)

(E)

p(r) (-)

(D)

p(r) (-)


1.25

1.75 2.0 2.25

0

0

20 40 60 80 r (Å)

0

20 40 60 80 r (Å)

20 40 60 80 r (Å)

2.50 2.75

1

1

1

0.1

q (Å-1)

0.1

1

q (Å-1)

0.1

1

q (Å-1)

1

q2I(q) (arb. units)

Figure 7 (A)

(C)

(B)

GdmCl conc. (M) 0 0.25 0.50 0.75 1.0 1.25 1.50

q2I(q) (arb. units)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 37

(D)

(F)

(E)

1.75 2.0 2.25 2.50 2.75

0.1

0.3 0.5 q (Å-1)

0.7

0.1

0.3 0.5 q (Å-1)

0.7

0.1


0.3 0.5 q (Å-1)

0.7

Page 31 of 37

Figure 8 30.0

g

R (Å)

27.5

sugar-free 5% trehalose 5% sucrose 5% glucose 10% trehalose 10% sucrose 10% glucose 10% fructose

25.0 22.5 20.0 17.5 15.0 0

0.5

1

1.5

2

2.5

3

GdmCl conc. (M)

Figure 9

molar fraction of native state (-)


1 0.8 0.6 0.4

(A)

0.2 0

sugar-free

1

5% trehalose 5% sucrose

0.8

5% glucose

0.6

10% trehalose

0.4

(B)

10% sucrose

0.2 10% glucose 0

10% fructose

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.8 0.6 0.4

(C)

0.2 0 0

0.5

1

1.5

2

2.5

3

GdmCl conc. (M)



Figure 10


(A)

GdmCl conc. (M) 0 0.75 1.25 1.50 1.75 2.00

1


(B)

1

0.01

0.1

1 -1

q (Å )

Figure 11 27.5 25.0

g

R (Å)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

22.5 20.0 17.5 15.0 12.5 0

0.5

1

1.5

2

GdmCl conc. (M)


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Page 33 of 37

Abs/Abs280 (-)

(A)

Abs/Abs280 (-)

Figure 12

(B)

Abs/Abs280 (-)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(C)

250

300

350

400

450

500

550

wavelength (nm)



Figure 13

Abs409/Abs280 (-)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

sugar free 20 % trehalose 20 % sucrose

0

0.5

1

1.5

2

2.5

3

GdmCl conc. (M)


Page 34 of 37

Page 35 of 37

Figure 14


(A)

1.2

1.3

1.4

1.5

1

temp. (°C) 25.0 30.0


35.0 40.0

(B)

45.0 50.0 55.0 60.0 1.2

1.3

1.4

1.5

65.0 70.0

1

72.5 75.0 77.5 80.0 82.5

(C) logI(q) (arb. units)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


85.0

1.2

1.3

1.4

1.5

1

0.1

1 -1

q (Å )



Figure 15

molar fraction of low temp.(25°C) state (-)

sugar free 10.4 % trehalose 10.4 % sucrose 10.4 % glucose 10.4 % fructose

21.7 % trehalose 21.7 % sucrose 21.7 % glucose 21.7 % fructose

1 0.8 0.6 0.4 0.2

(A)

(B)

(C)

(D)

0

molar fraction of low temp.(25°C) state (-)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 37

1 0.8 0.6 0.4 0.2 0 25

35

45 55 65 Temp. (°C)

75

85 25

35

45

55

65

75

Temp. (°C)


85

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Sugar-Mediated Stabilization of Protein against Chemical or Thermal

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