Fusion of amyloid beta with ferritin yields an isolated oligomeric beta-sheet-rich aggregate inside the ferritin cage.

Alzheimer's disease is a severe brain condition caused by the formation of amyloid plaques composed of amyloid beta (Aß) peptides. These peptides form oligomers, protofibrils, and fibrils before deposition into amyloid plaques. Among these intermediates, Aß oligomers (AßOs) were found to be the most toxic and therefore an appealing target for drug development and understanding their role in the disease. However, precise isolation and characterization of AßOs have proven challenging because AßOs tend to aggregate and form heterogeneous mixtures in solution. As a solution, we genetically fused the Aß peptide with a ferritin monomer. Such fusion allowed the encapsulation of precisely 24 Aß peptides inside the 24-mer ferritin cage. Using high-speed atomic force microscopy (HS-AFM), we disassembled ferritin and directly visualized the Aß core enclosed within the cage. The thioflavin-T assay (ThT) and attenuated total reflection infrared spectroscopy (ATR-IR) revealed the presence of a ß-sheet structure in the encapsulated oligomeric aggregate. Gallic acid, an amyloid inhibitor, can inhibit the fluorescence of ThT bound AßOs. Our approach represents a significant advancement in the isolation and characterization of ß-sheet rich AßOs and is expected to be useful for future studies of other disordered peptides such as α-synuclein and tau.

Mechanistic Modeling of Amyloid Oligomer and Protofibril Formation in Bovine Insulin.

Early phase of amyloid formation, where prefibrillar aggregates such as oligomers and protofibrils are often observed, is crucial for understanding pathogenesis. However, the detailed mechanisms of their formation have been difficult to elucidate because they tend to form transiently and heterogeneously. Here, we found that bovine insulin protofibril formation proceeds in a monodisperse manner, which allowed us to characterize the detailed early aggregation process by light scattering in combination with thioflavin T fluorescence and Fourier transform infrared spectroscopy. The protofibril formation was specific to bovine insulin, whereas no significant aggregation was observed in human insulin. The kinetic analysis combining static and dynamic light scattering data revealed that the protofibril formation process in bovine insulin can be divided into two steps based on fractal dimension. When modeling the experimental data based on Smoluchowski aggregation kinetics, an aggregation scheme consisting of initial fractal aggregation forming spherical oligomers and their subsequent end-to-end association forming protofibrils was clarified. Furthermore, the analysis of temperature and salt concentration dependencies showed that the end-to-end association is the rate-limiting step, involving dehydration. The established model for protofibril formation, wherein oligomers are incorporated as a precursor, provides insight into the molecular mechanism by which protein molecules assemble during the early stage of amyloid formation.

Oligomerization and aggregation of NAP-22 with several metal ions.

Metal ions participate in various biochemical processes such as electron transport chain, gene transcription, and enzymatic reactions. Furthermore, the aggregation promoting effect of several metal ions on neuronal proteins such as prion, tau, Aß peptide, and α-synuclein, has been reported. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the detergent-resistant membrane microdomain fraction of the neuronal cell membrane. Previously, we showed oligomer formation of NAP-22 in the presence of several phospholipids and fatty acids. In this study, we found the aggregation of NAP-22 by FeCl2, FeCl3, and AlCl3 using native-PAGE. Oligomer or aggregate formation of NAP-22 by ZnCl2 or CuSO4 was shown with SDS-PAGE after cross-linking with glutaraldehyde. Morphological analysis with electron microscopy revealed the formation of large aggregates composed of small annular oligomers in the presence of FeCl3, AlCl3, or CuSO4. In case of FeCl2 or ZnCl2, instead of large aggregates, scattered annular and globular oligomers were observed. Interestingly, metal ion induced aggregation of NAP-22 was inhibited by several coenzymes such as NADP+, NADPH, or thiamine pyrophosphate. Since NAP-22 is highly expressed in the presynaptic region of the synapse, this result suggests the participation of metal ions not only on the protein and membrane dynamics at the presynaptic region, but also on the metabolic regulation though the interaction with coenzymes.

Hyper Thermostability and Liquid-Crystal-Like Properties of Designed α-Helical Peptide Nanofibers.

Structural and thermodynamic transitions of artificially designed α-helical nanofibers were investigated using eight peptide variants, including four peptides with amide-modified carboxyl termini (CB peptides) and four unmodified peptides (CF peptides). Temperature-dependent circular dichroism spectroscopy and differential scanning calorimetry showed that CB peptides exhibit thermostability up to 50 °C higher than CF peptides. As a result, one of the denaturation temperatures approached nearly 130 °C, which is exceptionally high for a biomacromolecule. Thermodynamic analysis and microscopy observations also showed that CB peptides undergo a thermal transition similar to the phase transition in liquid crystals. In addition, one of the peptides showed a sharp and highly cooperative transition with a small enthalpy change at around 25 °C, which was ascribed to a giga-bundle burst of the molecular assembly. These macroscopic changes in the thermostability and crystallinity of CB peptides may be attributed to an increased amphiphilicity of the molecule in the direction of the helix axis, originating from the microscopic modification of the carboxyl-terminus.

Variations in the Protein Hydration and Hydrogen-Bond Network of Water Molecules Induced by the Changes in the Secondary Structures of Proteins Studied through Near-Infrared Spectroscopy.

This study investigated how the secondary structural changes of proteins in aqueous solutions affect their hydration and the hydrogen-bond network of water molecules using near-infrared (NIR) spectroscopy. The aqueous solutions of three types of proteins, i.e., ovalbumin, ß-lactoglobulin, and bovine serum albumin, were denatured by heating, and changes in the NIR bands of water reflecting the states of hydrogen bonds induced via protein secondary structural changes were investigated. On heating, the intermolecular hydrogen bonds between water molecules as well as between water and protein molecules were broken, and protein molecules were no longer strongly bound by the surrounding water molecules. Consequently, the denaturation was observed to proceed depending on the thermodynamic properties of the proteins. When the aqueous solutions of proteins were cooled after denaturation, the hydrogen-bond network was reformed. However, the state of protein hydration was changed owing to the secondary structural changes of proteins, and the variation patterns were different depending on the protein species. These changes in protein hydration may be derived from the differences in the surface charges of proteins. The elucidation of the mechanism of protein hydration and the formation of the hydrogen-bond network of water molecules will afford a comprehensive understanding of the protein functioning and dysfunctioning derived from the structural changes in proteins.

Detection of Fibril Nucleation in Micrometer-Sized Protein Condensates and Suppression of Sup35NM Fibril Nucleation by Liquid-Liquid Phase Separation.

Elucidating the link between amyloid fibril formation and liquid-liquid phase separation (LLPS) is crucial in understanding the pathologies of various intractable human diseases. However, the effect of condensed protein droplets generated by LLPS on nucleation (the initial step of amyloid formation) remains unclear because of the lack of available quantitative analysis techniques. This study aimed to develop a measurement method for the amyloid droplet nucleation rate based on image analysis. We developed a method to fix micrometer-sized droplets in gel for long-term observation of protein droplets with known droplet volumes. By combining this method with image analysis, we determined the nucleation dynamics in droplets of a prion disease model protein, Sup35NM, at the single-event level. We found that the nucleation was unexpectedly suppressed by LLPS above the critical concentration (C*) and enhanced below C*. We also revealed that the lag time in the Thioflavin T assay, a semi-quantitative parameter of amyloid nucleation rate, does not necessarily reflect nucleation tendencies in droplets. Our results suggest that LLPS can suppress amyloid nucleation, contrary to the conventional hypothesis that LLPS enhances it. We believe that the proposed quantitative analytical method will provide insights into the role of LLPS from a pathological perspective.

Tracking the Structural Development of Amyloid Precursors in the Insulin B Chain and the Inhibition Effect by Fibrinogen.

Amyloid fibrils are abnormal protein aggregates associated with several amyloidoses and neurodegenerative diseases. Prefibrillar intermediates, which emerge before amyloid fibril formation, play an important role in structure formation. Therefore, to prevent fibril formation, the mechanisms underpinning the structural development of prefibrillar intermediates must be elucidated. An insulin-derived peptide, the insulin B chain, is known for its stable accumulation of prefibrillar intermediates. In this study, the structural development of B chain prefibrillar intermediates and their inhibition by fibrinogen (Fg) were monitored by transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) combined with solid-state nuclear magnetic resonance spectroscopy (NMR) and size exclusion chromatography. TEM images obtained in a time-lapse manner demonstrated that prefibrillar intermediates were wavy rod-like structures emerging from initial non-rod-like aggregates, and their bundling was responsible for protofilament formation. Time-resolved SAXS revealed that the prefibrillar intermediates became thicker and longer as a function of time. Solid-state NMR measurement suggested a ß-sheet formation around Ala14 residue was crucial for the structural conversion from prefibrillar intermediates to amyloid fibril. These observations suggested that prefibrillar intermediates serve as reaction fields for amyloid nucleation and its structural propagation. Time-resolved SAXS also demonstrated that Fg prevented elongation of the prefibrillar intermediates by forming specific complexes together, which implied that regulation of the length of prefibrillar intermediates upon Fg binding was the factor suppressing the prefibrillar intermediate elongation. The fibril formation mechanism and the inhibition strategy found in this study will be helpful in seeking appropriate methods against amyloid-related diseases.

Multistep growth of amyloid intermediates and its inhibition toward exploring therapeutic way: A case study using insulin B chain and fibrinogen.

It is crucial to understand the mechanism of amyloid fibril formation for the development of the therapeutic ways against amyloidoses and neurodegenerative diseases. Prefibrillar intermediates, which emerge prior to the fibril formation, seem to play a key role to the occurrence of nuclei of amyloid fibrils. We have focused on an insulin-derived peptide, B chain, to precisely clarify the mechanism of the fibril formation via prefibrillar intermediates. Various kinds of methods such as circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering, and atomic force microscopy were employed to track the structural changes in prefibrillar intermediates. The prefibrillar intermediates possessing rod-shaped structures elongated as a function of time, which led to fibril formation. We have also found that a blood clotting protein, fibrinogen, inhibits the amyloid fibril formation of B chain. This was caused by the stabilization of prefibrillar intermediates and thus the suppression of their elongation by fibrinogen. These findings have not only shed light on detailed mechanisms about how prefibrillar intermediates convert to the amyloid fibril, but also demonstrated that inhibiting the structural development of prefibrillar intermediates is an effective strategy to develop therapeutic ways against amyloid-related diseases. This review article is an extended version of the Japanese article, Observing Development of Amyloid Prefibrillar Intermediates and their Interaction with Chaperones for Inhibiting the Fibril Formation, published in SEIBUTSU BUTSURI Vol. 61, p. 236-239 (2021).

Pathway Dependence of the Formation and Development of Prefibrillar Aggregates in Insulin B Chain.

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.

Current Understanding of the Structure, Stability and Dynamic Properties of Amyloid Fibrils.

Amyloid fibrils are supramolecular protein assemblies represented by a cross-ß structure and fibrous morphology, whose structural architecture has been previously investigated. While amyloid fibrils are basically a main-chain-dominated structure consisting of a backbone of hydrogen bonds, side-chain interactions also play an important role in determining their detailed structures and physicochemical properties. In amyloid fibrils comprising short peptide segments, a steric zipper where a pair of ß-sheets with side chains interdigitate tightly is found as a fundamental motif. In amyloid fibrils comprising longer polypeptides, each polypeptide chain folds into a planar structure composed of several ß-strands linked by turns or loops, and the steric zippers are formed locally to stabilize the structure. Multiple segments capable of forming steric zippers are contained within a single protein molecule in many cases, and polymorphism appears as a result of the diverse regions and counterparts of the steric zippers. Furthermore, the ß-solenoid structure, where the polypeptide chain folds in a solenoid shape with side chains packed inside, is recognized as another important amyloid motif. While side-chain interactions are primarily achieved by non-polar residues in disease-related amyloid fibrils, the participation of hydrophilic and charged residues is prominent in functional amyloids, which often leads to spatiotemporally controlled fibrillation, high reversibility, and the formation of labile amyloids with kinked backbone topology. Achieving precise control of the side-chain interactions within amyloid structures will open up a new horizon for designing useful amyloid-based nanomaterials.

Breakdown of supersaturation barrier links protein folding to amyloid formation.

The thermodynamic hypothesis of protein folding, known as the "Anfinsen's dogma" states that the native structure of a protein represents a free energy minimum determined by the amino acid sequence. However, inconsistent with the Anfinsen's dogma, globular proteins can misfold to form amyloid fibrils, which are ordered aggregates associated with diseases such as Alzheimer's and Parkinson's diseases. Here, we present a general concept for the link between folding and misfolding. We tested the accessibility of the amyloid state for various proteins upon heating and agitation. Many of them showed Anfinsen-like reversible unfolding upon heating, but formed amyloid fibrils upon agitation at high temperatures. We show that folding and amyloid formation are separated by the supersaturation barrier of a protein. Its breakdown is required to shift the protein to the amyloid pathway. Thus, the breakdown of supersaturation links the Anfinsen's intramolecular folding universe and the intermolecular misfolding universe.

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity.

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis-Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.

Multistep Changes in Amyloid Structure Induced by Cross-Seeding on a Rugged Energy Landscape.

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes, including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures, depending on species, each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new type of amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin seeds. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition analysis of the ultraviolet-visible absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.

Iodine staining as a useful probe for distinguishing insulin amyloid polymorphs.

It is recently suggested that amyloid polymorphism, i.e., structural diversity of amyloid fibrils, has a deep relationship with pathology. However, its prompt recognition is almost halted due to insufficiency of analytical methods for detecting polymorphism of amyloid fibrils sensitively and quickly. Here, we propose that iodine staining, a historically known reaction that was firstly found by Virchow, can be used as a method for distinguishing amyloid polymorphs. When insulin fibrils were prepared and iodine-stained, they exhibited different colors depending on polymorphs. Each of the colors was inherited to daughter fibrils by seeding reactions. The colors were fundamentally represented as a sum of three absorption bands in visible region between 400 and 750 nm, and the bands showed different titration curves against iodine, suggesting that there are three specific iodine binding sites. The analysis of resonance Raman spectra and polarization microscope suggested that several polyiodide ions composed of I3- and/or I5- were formed on the grooves or the edges of ß-sheets. It was concluded that the polyiodide species and conformations formed are sensitive to surface structure of amyloid fibrils, and the resultant differences in color will be useful for detecting polymorphism in a wide range of diagnostic samples.

Exploration of Insulin Amyloid Polymorphism Using Raman Spectroscopy and Imaging.

We aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na2SO4) by heating at 60, 70, 80, and 90°C. The intensity ratio of two Raman bands at 1672 and 1657 cm-1 due to antiparallel ß-sheet and α-helix structures, respectively, was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the ß-sheet structure was higher in the case with salts, especially at a higher temperature with Na2SO4. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond (∼514 cm-1) and the ratio of the tyrosine doublet I850/I826 were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis. Especially, amyloid fibrils with Na2SO4 media formed the gauche-gauche-gauche conformation of disulfide bond at a higher rate, but without any salts, the gauche-gauche-gauche conformation was partially transformed into the gauche-gauche-trans conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three- dimensional structural differences of amyloid polymorphisms. These results demonstrate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.

Theoretical Modeling of Electronic Structures of Polyiodide Species Included in α-Cyclodextrin.

The molecular mechanism of blue color formation in an iodine-starch reaction is studied by employing the iodine-α-cyclodextrin (α-CD) complex as a practical model system that resembles the structural properties of the blue amylose-iodine complex. To this end, we construct, using the quantum chemistry method, a molecular model of the complex (I5-/Li+/2α-CD) that consists of one I5-, two molecules of α-CD, and a lithium cation, and this model is employed as a basic unit in constructing the structural models of polyiodide ions (I5-)n. The initial structure in the geometry optimization is adopted from the α-CD-iodine complex structure obtained from the X-ray crystallography study. The structural models of (I5-)n are built by adding the basic unit n times along the crystal axis and by optimizing the structure using quantum mechanics/molecular mechanics (QM (iodine)/MM (α-CD)) calculations. The electronic absorption spectra of the resulting model structures are calculated by time-dependent density functional theory (TD-DFT). We find that I5- acts as a basic unit of coloration in the visible region. The visible color originates from the electronic transition within the I5- molecule, and any charge transfer between the I5- ion and either of α-CD or a coexisting counter cation is not involved. We also reveal that the electronic transitions of (I5-)n are delocalized, which accounts for the well-known observation that the color of the iodine-starch reaction becomes bluish with an increase in the chain length of amylose. Furthermore, the preresonance Raman spectra calculated from the model suggest that the vibrational motions are localized in the I5- subunit dominantly. A comparison between an experimental absorption spectrum feature of the α-CD-iodine complex and the calculated ones of (I5-)n ions with various n values suggests that (I5-)4 polyiodide ions tend to be populated dominantly in the α-CD-iodine complex under aqueous conditions.

Structural Insights into the Inhibition of Amyloid Fibril Formation by Fibrinogen via Interaction with Prefibrillar Intermediates.

Abnormal protein aggregation tends to result in the formation of ß-sheet rich amyloid fibrils, which are related to various kinds of amyloidoses and neurodegenerative diseases. The susceptibility to aggregation of protein molecules is dealt with by proteostasis in living systems, in which molecular chaperones play an important role. Recently, several secreted proteins have been examined as extracellular chaperones with a potency to suppress the formation of amyloid fibrils, although the whole picture that includes their inhibition mechanisms is not yet understood. In this study, we investigated the inhibitory effect of fibrinogen (Fg), one of the extracellular proteins identified as a potential member of the group of chaperones, on fibril formation. Insulin B chain was used as an amyloid formation model system because its prefibrillar intermediate species in the nucleation phase were well characterized. We revealed that Fg efficiently inhibited amyloid fibril formation via a direct interaction with the surface of the prefibrillar intermediates. Small-angle X-ray scattering experiments and a stoichiometry analysis suggested a structural model in which the surface of the rod-shaped prefibrillar intermediates is surrounded by Fg molecules. From such a specific manner of interactions, we propose that the role of Fg is to disturb fibril growth by confining the nuclei even when the nucleation occurs inside the prefibrillar intermediate. The structural property of the B-chain intermediates complexed with Fg would provide insights into the general principles of the functions of chaperones and other potential chaperone-like proteins involved in amyloid-related diseases.

9-Aryl-3-aminocarbazole as an Environment- and Stimuli-Sensitive Fluorogen and Applications in Lipid Droplet Imaging.

Environment-sensitive luminophoric molecules have played an important role in the fields of smart materials, sensing, and bioimaging. In this study, it was demonstrated that depending on the substituents, 9-aryl-3-aminocarbazoles can display aggregation-induced emission and solvatofluorochromism, and the operating mechanism was clarified. The application of these compounds to lipid droplet imaging and fluorescent probes for cysteamine was demonstrated.

A specific form of prefibrillar aggregates that functions as a precursor of amyloid nucleation.

Non-fibrillar protein aggregates that appear in the earlier stages of amyloid fibril formation are sometimes considered to play a key role in amyloid nucleation; however, the structural features of these aggregates currently remain unclear. We herein identified a characteristic pathway of fibril formation by human insulin B chain, in which two major species of prefibrillar aggregates were identified. Based on the time-resolved tracking of this pathway with far-UV circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and 1H-NMR spectroscopy, the first prefibrillar aggregate with a hydrodynamic diameter of approximately 70 nm accumulated concomitantly with the formation of a ß-sheet structure, and the size further evolved to 130 nm with an additional structural development. These prefibrillar aggregates were metastable and survived at least 24 hours as long as they were maintained under quiescent conditions. The energy barrier for nucleation was overcome by shaking or even by applying a single short ultrasonic pulse. Furthermore, an investigation where nucleation efficiency was monitored by fibrillation rates with varying the timing of the ultrasonic-pulse treatment revealed that the second prefibrillar aggregate specifically produced amyloid nuclei. These results suggest that the second form of the prefibrillar aggregates acts as a direct precursor for the amyloid nucleation.

Effect of Temperature and Hydration Level on Purple Membrane Dynamics Studied Using Broadband Dielectric Spectroscopy from Sub-GHz to THz Regions.

To investigate the effects of temperature and hydration on the dynamics of purple membrane (PM), we measured the broadband complex dielectric spectra from 0.5 GHz to 2.3 THz using a vector network analyzer and terahertz time-domain spectroscopy from 233 to 293 K. In the lower temperature region down to 83 K, the complex dielectric spectra in the THz region were also obtained. The complex dielectric spectra were analyzed through curve fitting using several model functions. We found that the hydrated states of one relaxational mode, which was assigned as the coupled motion of water molecules with the PM surface, began to overlap with the THz region at approximately 230 K. On the other hand, the relaxational mode was not observed for the dehydrated state. On the basis of this result, we conclude that the protein-dynamical-transition-like behavior in the THz region is due to the onset of the overlap of the relaxational mode with the THz region. Temperature hysteresis was observed in the dielectric spectrum at 263 K when the hydration level was high. It is suggested that the hydration water behaves similarly to supercooled liquid at that temperature. The third hydration layer may be partly formed to observe such a phenomenon. We also found that the relaxation time is slower than that of a globular protein, lysozyme, and the microscopic environment in the vicinity of the PM surface is suggested to be more heterogeneous than lysozyme. It is proposed that the spectral overlap of the relaxational mode and the low-frequency vibrational mode is necessary for the large conformational change of protein.