Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610–1,630 cm−1, characteristic of a β-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a β-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620 cm−1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70–90 % of the native form. Both irradiations at 1,535 cm−1(amide II band) and 1,240 cm−1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000 cm−1 afforded only about 60 % recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.

A mid-infrared free-electron laser (FEL) is operated as a pulsed and linearly polarized laser with tunable wavelengths within infrared region. Although the FEL can ablate soft tissues with minimum collateral damage in surgery, the potential of FEL for dissecting protein aggregates is not fully understood. Protein aggregates such as amyloid fibrils are in some cases involved in serious diseases. In our previous study, we showed that amyloid-like lysozyme fibrils could be disaggregated into the native form with FEL irradiation specifically tuned to the amide I band (1,620 cm−1). Here, we show further evidence for the FEL-mediated disaggregation of amyloid-like fibrils using insulin fibrils. Insulin fibrils were prepared in acidic solution and irradiated by the FEL, which was tuned to either 1,620 or 2,000 cm−1 prior to the experiment. The Fourier transform infrared spectroscopy (FT-IR) spectrum after irradiation with the FEL at 1,620 cm−1 indicated that the broad peak (1,630–1,660 cm−1) became almost a single peak (1,652 cm−1), and the β-sheet content was reduced to 25 from 40 % in the fibrils, while that following the irradiation at 2,000 cm−1 remained at 38 %. The Congo Red assay as well as transmission electron microscopy observation confirmed that the number of fibrils was reduced by FEL irradiation at the amide I band. Size-exclusion chromatography analysis indicated that the disaggregated form of fibrils was the monomeric form. These results confirm that FEL irradiation at the amide I band can dissect amyloid-like protein fibrils into the monomeric form in vitro.

No abstract

Amyloid fibrils are deposited in various tissues in the body, and are linked to the putative causes of serious diseases such as amyloidosis. Although the conditions of the disease would be expected to improve if the fibril structure could be destroyed, the aggregated structure is stable under physiological conditions. Recently, we found that the amyloid fibrils of lysozyme could be refolded into their active form by using a mid-infrared free-electron laser (MIR-FEL) tuned to the amide I band (corresponding to the C=O stretch vibration), with the MIR-FEL having specific oscillation characteristics of a picosecond pulse structure, a tunable wavelength within mid-infrared frequencies, and high photon density. In the study, we tested the usability of the FEL for dissociation of aggregates of pathological amyloid fibrils by using a short peptide of human thyroid hormone. The fibrils (after being placed on a glass slide) were irradiated using the FEL tuned to the amide I band (1644 cm −1), and those in situ were analyzed by Congo-Red assay, scanning-electron microscopy, and transmission-electron microscopy. All of the results obtained using these microscopic analyses indicated that the amyloid fibril formation was considerably decreased by FEL irradiation. Moreover, upon irradiation, a strong fibril peak at the amide I band in the infrared spectrum was transformed into a broad peak. These results imply that the β-sheet-rich structure of the amyloid fibrils changed into non-ordered or unspecified structures after the FEL irradiation. This FEL irradiation system, combined with various analytical methods, shows promise for the dissociation of amyloid aggregates.

Amyloid fibrils are widely recognized as a cause of serious amyloidosis such as Alzheimer's disease. Although dissociation of amyloid fibril aggregates is expected to lead to a decrease in the toxicity of the fibrils in cells, the fibril structure is robust under physiological conditions. We have irradiated amyloid fibrils with a free-electron laser (FEL) tuned to mid-infrared frequencies to induce dissociation of the aggregates into monomer forms. We have previously succeeded in dissociating fibril structures of a short peptide of the thyroid hormone by tuning the oscillation frequency to the amide I band, but the detailed structural changes of the peptide have not yet been determined at a high spatial resolution. Synchrotron-radiation infrared microscopy (SR-IRM) is a powerful tool for in situ analysis of minute structural changes of various materials, and in this study, the feasibility of SR-IRM for analyzing the microscopic conformational changes of amyloid fibrils after FEL irradiation was investigated. Reflection spectra of the amyloid fibril surface showed that the amide I peaks shifted to higher wave numbers after the FEL irradiation, indicating that the initial β-sheet-rich structure transformed into a mixture of non-ordered and turn-like peptide conformations. This result demonstrates that conformational changes of the fibril structure after the FEL irradiation can be observed at a high spatial resolution using SR-IRM analysis and the FEL irradiation system can be useful for dissociation of amyloid aggregates.

Structure of amyloid β (Aβ) fibrils is rigidly stacked by β-sheet conformation, and the fibril state of Aβ is profoundly related to pathogenesis of Alzheimer's disease (AD). Although mid-infrared light has been used for various biological researches, it has not yet been known whether the infrared light changes the fibril structure of Aβ. In this study, we tested the effect of irradiation of intense mid-infrared light from a free-electron laser (FEL) targeting the amide bond on the reduction of β-sheet content in Aβ fibrils. The FEL reduced entire contents of proteins exhibiting β-sheet structure in brain sections from AD model mice, as shown by synchrotron-radiation infrared microscopy analysis. Since Aβ fibril absorbed a considerable FEL energy at amide I band (6.17 μm), we irradiated the FEL at 6.17 μm and found that β-sheet content of naked Aβ fibril was decreased using infrared microscopic analysis. Consistent with the decrease in the β-sheet content, Congo-red signal is decreased after the irradiation to Aβ fibril. Furthermore, electron microscopy analysis revealed that morphologies of the fibril and proto-fibril were largely changed after the irradiation. Thus, mid-infrared light dissociates β-sheet structure of Aβ fibrils, which justifies exploration of possible laser-based therapy for AD.

Fibrous peptides such as amyloid fibrils have various roles in biological system, e.g., as causal factor of serious amyloidosis in human and as functional regulator of cell formation in bacteria and eukaryotes. In addition, the fiber-type format is promising as biocompatible scaffold. Therefore, the dissolution method of peptide fibril is potentially useful at many scenes in medical and material fields: as reductive way of pathogenic amyloid, as modification technique of cell structure, and as fabrication tool of biomaterials. However, the fibril structure is generally difficult to be dissociated due to its rigid stacked conformation. Here, we propose a physical engineering technology using terahertz free electron laser (FEL) at far-infrared wavelengths from 70 to 80 μm. Infrared microscopy analysis of the irradiated fibril of calcitonin peptide as a model showed that β-sheet was decreased, and α-helix, turn, and others were increased, compared to those of the fibril before the FEL irradiation. Interestingly, the dissociative effect by the far-infrared laser was remarkable than that by the mid-infrared laser tuned to 6.1 μm that corresponds to amide I. In addition, simple heating at 363 K deformed the fibril state but increased the amount of β-sheet, which was contrast with the action by the FEL, and scanning-electron microscopy and Congo-red staining revealed that the fibril was collapsed power-dependently within a range from 25 to 900 mJ energies supplied with the FEL at 74 μm. It can be considered that irradiation of intense terahertz wave can dissociate fibrous conformation of peptide with little influence of thermal effect.

Aggregations of proteins are in many cases associated with neurodegenerative diseases such as Alzheimer's (AD). Small compounds capable of inhibiting protein aggregation are expected to be useful for not only in the treatment of disease but also in probing the structures of aggregated proteins. In previous studies using phage display, we found that arginine-rich short peptides consisting of four or seven amino acids bound to soluble 42-residue amyloid β (Aβ42) and inhibited globulomer (37/48 kDa oligomer) formation. In the present study, we searched for arginine-containing small molecules using the SciFinder searching service and tested their inhibitory activities against Aβ42 aggregation, by sodium dodecyl sulfate (SDS)-PAGE and thioflavine T binding assay. Commercially available Arg-Arg-7-amino-4-trifluoromethylcoumarin was found to exhibit remarkable inhibitory activities to the formation of the globulomer and the fibril of Aβ42. This chimera-type tri-peptide is expected to serve as the seed molecule of a potent inhibitor of the Aβ aggregation process.