Photon Factory Activity Report 2018 #36 (2019)

Effect of Trehalose on Protein Structure Stabilization

BL-10C/2017G698

Satoshi AJITO1 and Mitsuhiro HIRAI1*Graduate School of Science and Technology,
Gunma University,
4-2 Aramaki, Maebashi, Gunma 371-8510, Japan.
11 Introduction
It is well known that some organisms that haveconsiderable tolerances against extreme environmentalconditions, produce stress proteins and/or accumulate
sugars in their cells. This phenomenon is called
cryptobiosis, i.e., the ability of an organism to tolerateenvironmental changes without having to actively adapt tothem. In particular, trehalose has been drawing attention
relevant to cryptobiosis under external stress such as highor low temperature, drying, and osmotic pressure. The
action of trehalose to afford an organism to tolerateenvironmental changes has been thought to result from therestriction of the intra-and/or-inter-molecular movementby vitrification or from the replacement of water moleculeby trehalose. It has been pointed out that the structure ofwater is an important factor. By the complementary use ofsynchrotron radiation wide-angle X-ray scattering (SRWAXS) and small-angle neutron scattering (SANS), we
have studied the effect of trehalose and other sugars onprotein structure and its hydration-shell against thermaland chemical denaturation.
2 Experiment
The protein measured was myoglobin from horseskeletal muscle, purchased from SIGMA Chemical Co.
(USA) and used without further purification. Trehalose(crystalline di-hydrated powder) from HAYASHIBARA
were used as sugars. The buffer solvent used was 10 mM
HEPES (N-(2-hydroxymethyl) piperazine-N'-(2-ethanesulfonic acid)) at pH 7.1 (pD 6.7) at 50 mM NaCl. The
myoglobin solution with 5 % w/v and the trehalosesolutions with the concentrations from 10 to 60 % w/w
were prepared as the stock solutions. The protein and sugar
stock solutions were mixed by 1:4 volume ratio, and usedas sample solutions. Just before the scattering
measurements, these solutions were filtered to remove
some large aggregates by using a Millipore membranefilter. The final protein concentrations of the solutions
were determined by ultraviolet-visible spectrophotometry
measurements.
SR-WAXS measurements were done by using the theBL-10C spectrometer at KEK, Tsukuba, Japan. The X-ray
wavelength and the sample-to-detector distance were 1.55
Å, 23.9 cm at BL-10C. The X-ray scattering intensity wasrecorded by the PILATUS3 2M detector. The exposure
time was 30 seconds at BL-10C. The temperature of thesolutions contained in the sample cells was controlled by
the temperature controller mK2000 of INSTEC Co. While
the measurements, the sample solutions were slowly
oscillated to avoid some radiation damages.

3 Results and Discussion
Figure 1 depicts the temperature dependence of theWAXS curves of myoglobin, where (A): in pure solvent,(B): in 20 % w/w trehalose solution. The observed WAXS
curve in each q-region reflects the protein structure at adifferent 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. As we observed the whole
hierarchal structure, the stabilization effect of trehalose on
the protein structure can be clarified in detail. The results
have been already published [1-3].

Fig. 1: Temperature dependence of the WAXS curve ofmyoglobin. (A) In water solvent, (B) In 20 % w/w
trehalose. The insets enlarge the WAXS curves in thesecondary structure region. The arrows indicate the typicalfeatures in the scattering curve appearing at the initialprocess of amyloid transition, namely, the pleated-betasheet stacking and the helix-to-sheet (cross-beta-sheet)transition.
References[1] S. Ajito, M. Hirai et al. J. Phys. Chem. B 122, 8685
(2018).
[2] M. Hiai, S. Ajito et al., J. Phys. Chem. B 122, 11962(2018).
[3] S. Ajito, M. Hirai et al., Physica B: Condensed Matter
551, 249 (2018).
*mhirai@gunma-u.ac.jp