How  do  sugars  affect  protein  structure  and  its  stability?     Gunma  University  Mitsuhiro  Hirai,  Satoshi  Ajito  1. Introduction
Structural stability of proteins in aqueous solutions not only depends on physical environment such as temperatureand pressure but also on the presence of osmolytes. It has been known for a long time that protein denaturationand enzyme deactivation can be prevented by the addition of sugar or polyol. Several factors, such as specificbinding between protein and additives and changes in solvent viscosity, have been considered. Proteins arepreferentially hydrated in the presence of sugars, and extensive hydrogen bonding of water in the hydration shellaround proteins in the presence of sugars has been considered to be a major driving force toward their stabilizing
effect. However, the experimental basis for the discussion so far is mostly the studies using spectroscopic methods
such as densitometry, calorimetry, circular dichroism, NMR, etc. There are few studies utilizing X-ray or neutron
scattering method that is applicable to observe direct evidences for the effect of sugars on protein structure andhydration. From biological points of view, under external stress such as high or low temperature, drying, osmoticpressure, and so on, some organisms that have tolerances against extreme environment produce stress proteins
and/or accumulate sugars in cells. It has been pointed out that the structure of water is an important factor.
Therefore, we have studied the effect of glucose on protein structure and its hydration-shell by the complementary
use of small-angle neutron scattering (SANS) and synchrotron radiation wide-angle X-ray scattering (SR-WAXS).
The experimental results obtained by SANS and SR-WAXS can be discussed in detail using the theoreticalscattering function simulations.
2. Experiment
SANS measurements were carried out by using the BL15 TAIKAN spectrometer at MLF at J-PARC, Tokai,
Japan. The neutron wavelength was 1.0 – 7.8 Å. The sample solutions were contained in the quartz cells with 1mm or 2 mm path length. The exposure time was around 1 - 3 hours. In the SANS measurements, we employed
the inverse contrast variation method. This method using deuterated materials is known to avoid or minimize theartificial effect on the scattering curves caused by the presence of co-solute molecules. We have also donesynchrotron radiation wide-angle X-ray scattering (SR-WAXS) measurements by using the BL-10C spectrometerat KEK, Tsukuba, Japan. We have succeeded to observe only a protein structure by mostly diminishing the effectof mimic-cell environment. The protein measured was myoglobin from horse skeletal muscle purchased fromSIGMA Chemical Co. (USA). Non-deuterated glucose and 97%-atom-deuterated glucose from SIGMA were usedas sugars. All other chemicals used were of analytical grade. The buffer solvent used was 10 mM HEPES(N-(2-hydroxymethyl) piperazine-N'-(2-ethane-sulfonic acid)) at pH 7.0 (pD 6.6) at 50 mM NaCl. The
concentration of myoglobin was 2 % w/v, and the sugar concentration was varied from 0 to 30 % w/w.
3. Results
Figure 1 shows the observed neutron scattering curve depending on the concentration of the glucose mixture([h-glucose]/[d-glucose] = 0.706/0.294), where the insert shows the square-root of the zero-angle scattering
intensity, I(0)1/2, depending on the glucose concentration. As shown in the insert, the magnitude of I(0)1/2 kept
constant, indicating that the effect of the presence of glucose on the contrast is mostly eliminated. In Figure 6, thesolid lines show the theoretical scattering functions obtained by fitting experimental data using the CRYSON
program, where the number of harmonics we used for the calculations was 50. The CRYSON program was also1

developed by Svergun et al. to simulate or fit neutron scattering functions based on the same architecture andprinciple of the CRYSOL program. As shown in Figure 1, the CRYSON fitting has been done very effectively
since the contrast of the protein is constant at every glucose

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concentration

glucose conc. (% w/w)
Exp_0
Exp_10
Exp_20
Exp_30underthemeasurement condition. The

presentneutron

scattering-square values of the fitting inFigure 1 were in the range from 1.0 to 1.6. The theoreticalscattering functions describe well the experimental ones. In

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Figure 2 (A) shows the distance distribution function, p(r),25(-)

obtained from the observed SANS curves in Figure 6 by using
20Equation 1. The maximum diameter, Dmax, of the particle

I(0)

101

1/2

I (q) (arb. units)

103

Fit_0
Fit_10
Fit_20
Fit_30shows a slightly decreasing tendency from 47.8 Å to 44.7 Å.

150

100

1020

glucose conc (% w/w)

30

0.1

In Figure 2 (B), the contrast of the hydration-shell obtained by

1-1q (Å )the CRYSON fitting and the Rg value are shown. It clearly

Figure 1: SANS curve of myoglobin
Neutron scattering curve depending on theglucose concentration (100 % D2O, 10 mM
Hepes, 50 mM NaCl, pD 6.7 (= pH 7.1) at25 °C)). Glucose is the mixture of[h-glucose]/[d-glucose] = 70.6/29.4. The
insert shows the zero-angle scattering
intensity, I(0)1/2. The solid lines show thetheoretical scattering functions obtained byfitting experimental data using CRYSON
program.

indicates that the hydration shell density is almost constant in(A)hydration-shell contrast preserves the value of ~0.58 x1010
cm--2 (corresponding to ~9.1 % higher than the average
scattering-density of D2O) up to 30 % w/w glucose. The above
results strongly support the WAXS results that sugar molecules
are preferentially excluded from the hydration-shell region ofthe protein.
4. Conclusion

0 % glucose w/w
10 %p(r) (arb. units)

spite of the increase of the glucose concentration. The

The neutron scattering results using the inverse-contrast

20 %
30 %

variation method strongly support the preferential exclusion ofsugar and the preservation of the hydration-shell of the protein.
At the sugar concentration higher than ~25 % w/w, the present

0102030r (Å)

40results suggest the possibility of the preferential solvation of50
0.8(B)

0.7gR (Å)

14.5

0.6
0.5

14.0

0.4
0.3

13.5

0.2

hydration-shell contrast (1015.0

1000102030glucose conentration (% w/w)

-2

13.0cm )0.1

sugars that will be partially occurring on the protein surface.
From the methodological point of view, it should be noted thatthe complementary use of X-ray and neutron scattering would
have a high availability or superiority to afford us a direct
evidence of the effect of various osmolytes on protein structuresand on those hydration properties under molecular crowding
environments. The detailed results and discussion will beshown soon [1].

Figure 2: (A), distance distributionfunction;(B),contrastof
thehydration-shell evaluated by the fitting andthe experimental Rg values obtained from
Figure 1.
References[1] S. Ajito, M. Hirai et al., Physica B, doi.org/10.1016/j.physb.2018.03.040.
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