High-power Terahertz Waves for a Recycle System of Amyloid Fibrils

19 Recycling of persistent materials is one of most important subjects to be addressed towards the 20 sustainable society. Amyloid fibril is such a tough biomaterial that can be designed for various 21 industrial applications, and it is usually difficult to dissociate the once made fibrous conformation 22 due to the cross  -sheet stacks. We propose here a unique but versatile approach to handle the 23 fibril formation by using two-kinds of high-power terahertz waves. Lysozyme and  2- 24 microglobulin peptide fragment were employed as model samples, and those fibrils were clearly 25 disaggregated accompanied by decrease of  -sheets and increase of  -helices by the irradiation 26 of 5.3 THz free electron laser tuned to 56  m, as shown by infrared (IR) microscopy and scanning- 27 electron microscopy (SEM). In contrast, those fibrous conformations were reversely self- 28 associated by the irradiation of 0.42 THz wave tuned to 720  m from gyrotron, as shown by 29 optical and IR microscopies, SEM, and small-angle X-ray scattering. The overall reaction is 30 performed at room temperature within 30 min without external heating and high-pressures. 31 Therefore, amyloid fibrils can be dissociated and associated under the proper far-infrared 32 radiation conditions, which inspires a sustainable recycling system of fibrous biomaterials.

systems 19,20 , and functional nanofilms for microorganism adhesion and protein crystallization for 48 medical purposes 21,22 . Therefore, a versatile approach for structural control of the amyloid fibrils 49 will be useful for remodeling of the fibrous format in various biomaterial fields. However, the 50 cross -sheet stacking structure, that is common in amyloid fibrils, is hydrophobic and stable in 51 water, which makes it difficult to regulate the once made fibril freely 23,24 . Of course, organic 52 solvents such as dimethyl sulfoxide are known as the melting reagent, and fibril binding molecules 53 are explored for modulating the fibril formation [25][26][27] . Nonetheless, for the most part, it may be 54 8 around 5 K compared to the non-irradiation area. The low-resolution microscopy observation (Fig. 127 3b) showed that the fibrous aggregate was observed like black-brown colors in both lysozyme 128 and 2-microglobulin before the irradiation (0 mJ). When the irradiation power was increased 129 from 10 mJ (10 W with 1ms pulse duration) to 20 mJ (10 W with 2 ms pulse duration), the black-130 brown colors were apparently concentrated in both cases (white totted circles). The high-131 resolution electron microscopy observation showed that the fibril structure changed into more 132 solid aggregates by the irradiation at 20 mJ power in both samples (Fig. 3c). In case of lysozyme, 133 needle-like fibrils (several hundred nanometer in width, several micrometers in length) were 134 sparse without irradiation (-), and thick branch-like fibrils (one micrometer in width, several 135 micrometers in length) were increased with the irradiation (+). In case of 2-microglobulin, the 136 assemblies of many strings (several hundred nanometer in width, ten to twelve micrometers in 137 length) before the irradiation (-) were clearly changed into bundles like clay after the irradiation 138 (+). 139 Figure 3d shows results by infrared absorption spectral analysis. In case of lysozyme (upper, 140 left panel), the peak intensity at around 1620 cm -1 was apparently increased after the irradiation 141 (red) compared to that before irradiation (black). The protein structure analysis (right panel) 142 indicated that -sheet was increased, and -helix was decreased by the irradiation 44 . In case of 143 2-microglobulin (below, left panel), the peak intensity at 1623 cm -1 before irradiation (gray) was 9 obviously increased accompanied by increase of proportion of -sheet (right panel) after the 145 irradiation (blue). 146 The result by SAXS analysis was shown in Fig. 3e. In both cases, the inclination of the scattering 147 curve from 3 nm -1 to 9 nm -1 was larger after the irradiation (red) than that before irradiation (black). 148 This means that the shape of the aggregate was changed into the thick lamellar type 45 . In addition, 149 there can be observed a scattering peak at around 3.8 nm -1 in lysozyme (upper) and at 3.7 nm -1 in 150 2-microglobulin (lower) after irradiation. These peaks mean that a size (d) of layer of the fibril 151 is 1.65 nm and 1.69 nm, respectively. These values are quite larger than the typical size (0.9-1.0 152 nm) of the amyloid fibril 46 . 153 154

Recycle of fibrous biomaterials by the far-infrared radiation 155
The above all results suggested that the fibrous conformations of lysozyme and 2-156 microglobulin were dissociated by the THz-FEL and associated by the submillimeter wave from 157 gyrotron. This study implied that dissociation and association of amyloid fibrils can be performed 158 in one batch system by using terahertz waves properly at different wavelengths (Fig. 4). By using 159 both terahertz radiations continuously, the amyloid-base fiber biomaterials can be recycled 160 without denature of the protein backbone. This method requires no organic solvents, no external 161 heating, and no high pressures, which inspires that the electromagnetic waves at terahertz region 162 10 will become a green technology for the sustainable system of fibrous biomaterials. We 163 demonstrated that the submillimeter wave can promote the fibril formation of many kinds of 164 amyloid peptides (GNNQQNY, A1-40, SAA, and DFNKF in our previous study 47 , lysozyme and 165 2-microglobulin in the present paper), and in every case, -sheet conformation was dominated 166 and the sample was more aggregated than the pre-irradiation state. Nonetheless, the reformed 167 aggregate seems to be shapely larger and more rigid than the pre-irradiation state (Fig. 3c, e). 168 Therefore, it can be implied that the submillimeter wave from gyrotron will be a versatile tool for 169 remodeling of amyloid-base fiber biomaterials, and this method will be potentially applied for 170 modifying other fibrous materials such as cellulose nano-fibers 1-5 to improve the fibrous 171 characteristics such as the rigidness and the regularity. Previously, we reported that the THz-FEL can dissociate an amyloid fibril from calcitonin 173 DFNKF peptide 48 . Together with this prior study, it was revealed that several kinds of amyloid 174 fibrils can be dissociated by the THz-FEL. The tendency of decrease of -sheets by the irradiation 175 was varied dependent on the molecular size of amyloid: the proportion of the -sheet of DFNKF 176 was decreased from 40% to 10% 48 , that of 2-microglobulin was from 50% to 5%, and that of 177 lysozyme was from 45% to 20% (Fig. 2b). Therefore, smaller sized peptides (5 a.a. of DFNKF 178 and 11 a.a. of 2-microglobulin) may be easier to be dissociated than the larger sized protein (129 179 a.a. of lysozyme). Although the detailed mechanism is not clear at the present stage, it can be 180 11 considered that the dissociation process may be similar with the phenomenon under which a solid 181 aggregate is momentarily unraveled in boiling water. As one of experiments to investigate the 182 reaction mechanism, it can be planned to monitor the dissociation processes by using atomic force 183 microscopy in the presence of fibril-binding molecules 49 . This experiment will be a next 184 challenging theme. As a side application, THz-FEL can be applied to the amyloidosis therapy by 185 reducing pathogenic amyloid aggregates from tissues in surgical medicine, and to regulate the 186 growth of microorganisms by suppressing the biofilm formation related with amyloids in 187 synthetic biology. These themes are also fascinated as application studies of the terahertz rays. We proposed here that fibrous biomaterials can be recycled by using two-kinds of high-power far-191 infrared rays. One is THz-FEL that is accelerator-based picosecond pulse laser, and another is a 192 submillimeter wave from gyrotron. Lysozyme and 2-microglobulin peptide fragment were 193 employed as models, and THz-FEL tuned to 56 m can dissociate those stacking conformations 194 accompanied by decrease of -sheet and increase of -helix, and the submillimeter wave at 720 195 m can promote those fibrillations reversely, as revealed by infrared, electron, and optical 196 microscopies, and SAXS analyses. The total elapsed time is within 30 min, and those radiations 197 can be performed at room temperatures without any external heating and high-pressures.

12
Combination of these far-infrared radiations will be expected to contribute to a sustainable recycle 199 system of the fibrillar biomaterials in future. Lysozyme (from hen egg white) was purchased from Sigma-Aldrich (Tokyo, Japan). 2-204 Microglobulin (21-31, NFLNCYVSGFH) was purchased from PH-Japan (Hiroshima, Japan). 205 Acetic acid and sodium chloride were purchased from Wako Pure Chemical Industries (Osaka, 206 Japan). 207

Sample preparation 208
As for THz-FEL irradiation, lysozyme and 2-microglobulin were fibrillated as follows: 209 Lysozyme powder was dissolved in 20 % acetic acid (2.5 mg/mL) containing sodium chloride 210 (0.5 M), and the solution (1 mL) was incubated for 20 h at 37 ℃. The freeze-dried 2-211 microglobulin peptide was dissolved in dimethyl sulfoxide (40 mg/mL) and stocked at -20 ℃. 212 The portion of the stock solution was diluted by phosphate buffer saline (pH 7.5) containing 213 sodium chloride (100 mM) to be 2.0 mg/mL concentration and incubated at 37 ℃ for two days. 214 Those suspensions (each 10 L) were spotted on a stain less steel base for infrared microscopy or 215 13 those samples were irradiated by THz-FEL. 217 As for gyrotron experiments, samples were prepared as follows: Lysozyme was dissolved in 218 acidic water (150 L) as described above, and the solution was used for the irradiation experiment 219 without the subsequent thermal incubation. A portion of the stock solution of 2-microglobulin 220 peptide was diluted by the buffer as described above, and the solution was directly subjected to 221 the irradiation experiment without further incubation. 222

THz-FEL irradiation 223
The principle of the beam generation was briefly as follows: an FEL oscillation system consists 224 of an electron gun, sub-harmonic buncher, an accelerator tube, a periodic magnetic field (wiggler 225 in this case), and an optical cavity to amplify the FEL pulses. A small portion of the FEL pulses 226 in the cavity is extracted via a coupling hole that is 3 mm in diameter at the center of the upstream 227 resonant mirror. The wiggler is the Halbach-type magnetic field. The FEL beam is transported 228 through a concrete wall (3 m in thick) and through a diamond window of the monochromator to 229 the experimental room. The oscillation wavelengths were tuned to 56 or 70 m, and the amyloid 230 sample dried on a slide base was irradiated by the THz-FEL from the vertical direction at room 231 temperature with raster scan. Under this irradiation conditions, beam diameter was focused to 232 approximately 400 m by using a parabolic reflector, the irradiation area was 1 mm x 1mm square, 233 and the step scan length was set to 0.1 mm. 234 14

Gyrotron irradiation system 235
The gyrotron is a vacuum electron tube and the operation is based on a physical phenomenon 236 known as electron cyclotron maser instability. The structure is composed of an electron-optical 237 system based on a triode magnetron injection gun with a thermionic cathode that generates a 238 helical electron beam in the superconducting magnet, a cavity resonator for coupling the electron 239 beams with waves, an internal mode converter to adjust spatial distributions of oscillated waves, 240 an output vacuum window, and a water-cooled collector of the spent electron beams. The 241 submillimeter wave can be oscillated as Gaussian wave beam from the output vacuum window. 242 We used the Gyrotron FU CW GVIB far-infrared radiation system, which can expose samples to 243 a 420 GHz wave with 10 W power for 20 min. The radiation wavelength was 720 m, and the 244 pulse duration was 1 ms or 2 ms at 5 Hz repetition. The temperature increase of the sample during 245 the irradiation was monitored using a Testo 875 thermography camera (Testo). The amyloid 246 sample in aqueous solution (150 L) was put on the Eppendorf tube that is composed of 247 polypropylene and was irradiated at room temperature (ca. 25 °C) from vertical direction. 248

Terahertz spectroscopy 249
We used a far-infrared Fourier-transform spectrometer (IFS66v/S, Bruker) for the absorption 250 spectrum measurement at terahertz region. The sample powder was mixed with CsI powder and 251 pressed to form a mini-disk plate. The measurement was performed by transmission mode, and 252 the spectrum was recorded at 130-700 cm -1 with 32 scans using Mylar (polyester film) as beam 253 splitter. 254

Infrared microscopy 255
The mid-IR spectra were measured using IRT-7000 infrared microscope (Jasco Co, Tokyo, Japan) 256 and FT/IR-6100 spectrometer (Jasco Co., Tokyo, Japan). The dry surface of the sample film was 257 observed by 16x Cassegrain lens, and the infrared spectra were recorded by a reflection mode 258 with 64 scans and 4 cm -1 resolution. For analysis of protein secondary structure, we used IR-SSE 259 analytical software (Jasco Co., Tokyo, Japan) in which calibration curve data was prepared as a

Scanning-electron microscopy 266
We used FE-SEM Supra40 scanning electron microscope (Carl Zeiss). After the amyloid sample 267 was added on a glass slide base and dried under atmosphere, the slide base was fixed on a sample 268 holder by using conductive copper tape. The surface of the sample was observed using the 269 acceleration voltage at 5.0 kV. 270 16

Optical microscopy 271
The amyloid sample was added on a gold-coated slide base and dried under atmosphere. The 272 surface of the sample was observed using an Area PIII-FX microscope (SK-Electronics Co., LTD., 273 Kyoto, Japan) with a high-magnification object lens (×200-2000). Images were obtained using a 274 12 million-pixel CCD camera under the halogen lamp. Images of the sample surface were 275 obtained using Perfect Viewer 7 imaging software (SK-Electronics Co., LTD., Kyoto, Japan). 276

Small-angle X-ray scattering 277
X-ray scattering experiment was performed using the beamline BL8S3 in Aichi Synchrotron 278 Radiation Center (Aichi, Japan). As for lysozyme, the suspension containing the fibril was put on 279 a Teflon sheet (1mm in depth), and the sample was surrounded and encapsulated by using Kapton 280 tape that is made of polyimide film (TERAOKA SEISAKUSHO CO., LTD., Tokyo). The sample 281 cell was set at the vertical position against the X-ray direction. As for 2-macroglobulin, the 282 suspension was spotted on a cover glass and dried under atmosphere. The cover glass was set at 283 the vertical position against the X-ray direction. In both cases, the wavelength of X-ray was 0.15 284 nm and the sample-to-specimen length was 45 cm for measurements. The scattering patterns were 285 recorded by use of R-AXIS imaging plate (Rigaku, Japan). Each exposure time of X-ray was 600 286 s.

Supplementary Files
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