Photon Factory Activity Report 2022 #40 (2023)

BL-10C/2019G537

Structural Properties of Proteins Embedded in Sugar-Glass and/or Sugar-Rubber
Mitsuhiro HIRAI1, Shigeki ARAI2, Hiroshi NAKAGAWA3, and Hiroki IWASE4
1Graduate School of Science and Technology, Gunma University, Maebashi, Gunma 371-8510,
Japan.
2
National Institute for Quantum and Radiological Science and Technology, Inage, Chiba 263-8555,
Japan.
3
Materials Science Research Center, Tokai, Ibaraki 319-1195, Japan.
4Comprehensive Research Organization for Science and Society, Tokai, Ibaraki 319-1106, Japan
1 Introduction
Insects such as tardigrades, rotifers, and polypedilum
vanderplanki are well known as animal resistance inextreme environments (cryptobiosis "non-metabolicstate"), all of which are found in extreme environments
such as desiccation and freezing conditions. They survive
by accumulating sugar (trehalose) as a "compatible solute"that protects biological components and cell membranes
instead of water and stopping metabolic activity(hibernation, anhydrobiosis). It is also known thatamphibians can survive by creating a hyperglycemic state
when living conditions deteriorate such as low temperatureand dryness (extreme heat). Such environmental resistancemechanisms have been explained by the "glass statetheory" (protection of higher-order structures such as cell
membranes and proteins associated with vitrification bysugar) or the "water substitution theory" (direct hydrogenby sugar acting as a substitute for bound water). However,the details of the mechanism are still unknown because
there are few experimental studies at the molecular
structure level.
Recently, by the complementary use of X-ray andneutron scattering techniques, we have provided direct
evidence that the protein hydration (solvation) andstructural stability against chemical and thermal
denaturation significantly depend considerably on thesugar species and glycerol.1-3 The sugar and glycerol
molecules tend to be preferentially or weakly excluded
from the protein surface, which preserves the native
protein hydration shell; however, the preferential exclusion(preferential hydration) shifts gradually toward the nonpreferential solvation (replacement of the hydrated waterby sugar or glycerol) as the concentrations of thesemolecules increase. Owing to the protective action of thesemolecules on the protein hydration shell, the protein
structure is stabilized against chemical (guanidiniumchloride) and thermal denaturation. The protective action
depends on the sugar species.3 In addition, we also foundthat intermolecular interactions between sugar molecules
are essentially repulsive interactions. However, we haveobserved that the correlation distance between sugar
molecules varies significantly depending on the type ofsugar, and it depends on the hydration structure anddynamic properties of sugars.4

On the other hand, carbohydrates are one of the threemajor components of foods along with lipids and proteins.
At the same time, depending on changes in temperature
and water content during food processing，carbohydrates
undergo various physical property changes (crystallization,melting, glass transition, inclusion complex formation,etc.), which determines the quality and preservation offood. Currently, the importance of glass transition (Tg) isrecognized from the scientific point of view on freezing,storage, and thawing of foodstuffs. Therefore, if a
relatively easy storage temperature can be set by increasing
Tg by adding sugar, the storage period and quality can beextended. Tg varies depending on the type andconcentration of sugars. Thus, for long-term stable storage,
it is important to search for the optimum sugar vitrification
state conditions that can stably retain the structures ofproteins and lipids based on nanoscale-level structuralanalysis.
Thus, the purpose of this research is to elucidate thestructural characteristics of proteins trapped in extremely
low moisture conditions, such as sugar glassy and sugar
rubbery states, which are unresolved issues in the fields ofextremophile biology and food science.
2 Experiment
The proteins measured were horse skeletal muscle
myoglobin, egg white lysozyme, bovine serum albumin,horse hemoglobin, which were purchased from SigmaAldrich. We used four different types of sugars:
disaccharides (trehalose, sucrose) and monosaccharides(fructose, glucose). The proteins were dissolved in thewater solvent (0.5 mM HEPES, pH 7.1) to be theconcentration of 3 % w/v. The sugars were dissolved in thewater solvent to be the concentration of 30 % w/w. These
protein and sugar solutions were used as the stock solutions,respectively. After mixing the protein solution and thesugar solution at a ratio of 3/1, contained in the disc-shaped
sample cell, and dried in a constant temperature bath for 4days. The water contents of the samples served for thescattering measurement were evaluated from the weightmeasurement using an electronic balance. The watercontents of the samples were in the range from 3.2 % w/w
to 12 % w/w. The small- and wide-angle X-ray scattering(SWAXS) measurements were carried out by using theBL10C spectrometer at PF. The X-ray wavelength and thePhoton Factory Activity Report 2022 #40 (2023)

sample-to-detector distance was 1.5 Å for 24 cm, and 1.5
Å for 203 cm. The temperature of the he samples et in thesample cell holder was controlled by the model mK2000
temperature controller (Instec, Inc.).

[3] S. Ajito, M. Hirai et al., J. Phys. Chem. B 122, 11962(2018).
[4] M. Hirai, S. Ajito et al., J. Phys. Chem. B 5, 10815(2020).

3 Results and Discussion
Fig. 1 shows the WAXS curves of proteins embedded
sugar rubber at 20°C. The water contents in Fig.1 werearound 10% w/w. The scattering curves below q = ~0.4 Å1in Fig.1 reflect well characteristic scattering curves
depending on the structural properties of each protein at
native state. The values of the radius of gyration (Rg) ofproteins are known to depend on the difference between
the average scattering densities of the solute particle andthe solvent matrix, so-called contrast. The Rg values
estimated from Fig. 1 almost agrees with the theoreticalvalue considering contrast change due to excessive sugar
environment.

* mhirai@gunma-u.ac.jp

Fig. 1: WAXS curves of proteins embedded in different
types of sugar rubber at 20°C.
The difference in the scattering curves above q = ~0.5
Å-1 is attributable to the sugar matrix depending on thesugar type as we already reported in the results of highly
concentrated sugar solutions. The detailed results will beseen soon.
Acknowledgement
We thank the PF staff of small-angle X-ray scatteringgroup for supporting the for setting up the optical system
and assisting with the measurement.
References[1] M. Hirai, S. Ajito et al., Biophy. J. 115, 313 (2018).
[2] S. Ajito, M. Hirai et al., Physica B: Condensed Matter
551, 249 (2018).