Low pressure‐induced secondary structure transitions of regenerated silk fibroin in its wet film studied by time‐resolved infrared spectroscopy

The secondary structure transitions of regenerated silk fibroin (RSF) under different external perturbations have been studied extensively, except for pressure. In this work, time‐resolved infrared spectroscopy with the attenuated total reflectance (ATR) accessory was employed to follow the secondary structure transitions of RSF in its wet film under low pressure. It has been found that pressure alone is favorable only to the formation of β‐sheet structure. Under constant pressure there is an optimum amount of D₂O in the wet film (D₂O : film = 2:1) so as to provide the optimal condition for the reorganization of the secondary structure and to have the largest formation of β‐sheet structure. Under constant amount of D₂O and constant pressure, the secondary structure transitions of RSF in its wet film can be divided into three stages along with time. In the first stage, random coil, α‐helix, and β‐turn were quickly transformed into β‐sheet. In the second stage, random coil and β‐turn were relatively slowly transformed into β‐sheet and α‐helix, and the content of α‐helix was recovered to the value prior to the application of pressure. In the third and final stage, no measurable changes can be found for each secondary structure. This study may be helpful to understand the secondary structure changes of silk fibroin in silkworm's glands under hydrostatic pressure.     

Peptide contour length determines equilibrium secondary structure in protein‐analogous micelles

This work advances bottom‐up design of bioinspired materials built from peptide‐amphiphiles, which are a class of bioconjugates in which a biofunctional peptide is covalently attached to a hydrophobic moiety that drives self‐assembly in aqueous solution. Specifically, this work highlights the importance of peptide contour length in determining the equilibrium secondary structure of the peptide as well as the self‐assembled (i.e., micelle) geometry. Peptides used here repeat a seven‐amino acid sequence between one and four times to vary peptide contour length while maintaining similar peptide‐peptide interactions. Without a hydrophobic tail, these peptides all exhibit a combination of random coil and α‐helical structure. Upon self‐assembly in the crowded environment of a micellar corona, however, short peptides are prone to β‐sheet structure and cylindrical micelle geometry while longer peptides remain helical in spheroidal micelles. The transition to β‐sheets in short peptides is rapid, whereby amphiphiles first self‐assemble with α‐helical peptide structure, then transition to their equilibrium β‐sheet structure at a rate that depends on both temperature and ionic strength. These results identify peptide contour length as an important control over equilibrium peptide secondary structure and micelle geometry. Furthermore, the time‐dependent nature of the helix‐to‐sheet transition opens the door for shape‐changing bioinspired materials with tunable conversion rates. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 573–581, 2013.     

Geometrical principles of homomeric β‐barrels and β‐helices: Application to modeling amyloid protofilaments

Examples of homomeric β‐helices and β‐barrels have recently emerged. Here we generalize the theory for the shear number in β‐barrels to encompass β‐helices and homomeric structures. We introduce the concept of the “β‐strip,” the set of parallel or antiparallel neighboring strands, from which the whole helix can be generated giving it n‐fold rotational symmetry. In this context, the shear number is interpreted as the sum around the helix of the fixed register shift between neighboring identical β‐strips. Using this approach, we have derived relationships between helical width, pitch, angle between strand direction and helical axis, mass per length, register shift, and number of strands. The validity and unifying power of the method is demonstrated with known structures including α‐hemolysin, T4 phage spike, cylindrin, and the HET‐s(218‐289) prion. From reported dimensions measured by X‐ray fiber diffraction on amyloid fibrils, the relationships can be used to predict the register shift and the number of strands within amyloid protofilaments. This was used to construct models of transthyretin and Alzheimer β(40) amyloid protofilaments that comprise a single strip of in‐register β‐strands folded into a “β‐strip helix.” Results suggest both stabilization of an individual β‐strip helix and growth by addition of further β‐strip helices can involve the same pair of sequence segments associating with β‐sheet hydrogen bonding at the same register shift. This process would be aided by a repeat sequence. Hence, understanding how the register shift (as the distance between repeat sequences) relates to helical dimensions will be useful for nanotube design.     

Structural properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

Replica exchange molecular dynamics simulations (300 ns) were used to study the dimerization of amyloid β(1‐40) (Aβ(1‐40)) polypeptide. Configurational entropy calculations revealed that at physiological temperature (310 K, 37°C) dynamic dimers are formed by randomly docked monomers. Free energy of binding of the two chains to each other was ?93.56 ± 6.341 kJ mol^?1. Prevalence of random coil conformations was found for both chains with the exceptions of increased β‐sheet content from residues 16‐21 and 29‐32 of chain A and residues 15‐21 and 30‐33 of chain B with β‐turn/β‐bend conformations in both chains from residues 1‐16, 21‐29 of chain A, 1‐16, and 21‐29 of chain B. There is a mixed β‐turn/β‐sheet region from residues 33‐38 of both chains. Analysis of intra‐ and interchain residue distances shows that, although the individual chains are highly flexible, the dimer system stays in a loosely packed antiparallel β‐sheet configuration with contacts between residues 17‐21 of chain A with residues 17‐21 and 31‐36 of chain B as well as residues 31‐36 of chain A with residues 17‐21 and 31‐36 of chain B. Based on dihedral principal component analysis, the antiparallel β‐sheet‐loop‐β‐sheet conformational motif is favored for many low energy sampled conformations. Our results show that Aβ(1‐40) can form dynamic dimers in aqueous solution that have significant conformational flexibility and are stabilized by collapse of the central and C‐terminal hydrophobic cores with the expected β‐sheet‐loop‐β‐sheet conformational motif. Proteins 2017; 85:1024–1045. © 2017 Wiley Periodicals, Inc.     

Effect of EGCG On Fe(III)‐induced conformational transition of silk fibroin,a model of protein related to neurodegenerative diseases

The abnormal aggregation of amyloid proteins is reported to play a critical role in the etiology of neurodegenerative disorders. Studies have shown that excessive ferric irons are associated with the misfolding of amyloid proteins, and that (‐)‐epigallocatechin gallate (EGCG) is a good metallic ion chelator with inhibitory effect on the aggregation of amyloid proteins. EGCG has been thus considered as a potential drug candidate for the treatment of neurodegenerative diseases. However, the mechanism of action for EGCG in inhibition of aggregation of amyloid proteins is still remaining unclear. Silk fibroin (SF) shares similarities with amyloid proteins in some amino acid sequences and fibrillation kinetics. In this work, therefore, we used SF as a model of protein to investigate the effects of Fe(III) and EGCG on conformational transition by using turbidity assay, thioflavin T (ThT) fluorescence spectroscopy, Raman spectroscopy, and atomic force microscope (AFM). We demonstrated that low concentration of Fe(III) ions promoted the formation of β‐sheet conformers, while high concentration of Fe(III) ions inhibited further aggregation of SF. EGCG could significantly inhibit the conformational transition of SF when induced by Fe(III), and decrease the amount of β‐sheet conformers dose‐dependently. The findings provide important information regarding to EGCG as a potential agent for the prevention and treatment of neurodegenerative diseases. Fe(III) can accelerate the conformation transition of silk fibrion (SF) from random coil into β‐sheet, while (‐)‐epigallocatechin gallate (EGCG) inhibits Fe(III)‐induced β‐sheet aggregation of SF., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 100–107, 2016     

β‐sheet assembly in amyloidogenic glutamic acid nanostructures: Insights from X‐ray scattering and infrared nanospectroscopy

Glutamic acid–rich peptides are crucial to a variety of biological processes, including glutamatergic neurotransmission and immunological defense. Glutamic acid sequences often exhibit unusual organization into β₂‐type sheets, where bifurcated H bonds formed between glutamic acid side chains and NH in amide bonds on adjacent β‐strands play a paramount role for stabilizing the molecular assembly. Herein, we investigate the self‐assembly and supramolecular structure of simplified models consisting of alternating glutamic acid/phenylalanine residues. Small‐angle X‐ray scattering and atomic force microscopy show that the aggregation pathway is characterized by the formation of small oligomers, followed by coalescence into nanofibrils and nanotapes. Amyloidogenic features are further demonstrated through fiber X‐ray diffraction, which reveal molecular packing according to cross‐β patterns, where β‐strands appear perpendicularly oriented to the long axis of nanofibrils and nanotapes. Nanoscale infrared spectroscopy from individual nanoparticles on dried samples shows a remarkable decrease of β₂‐sheet content, accompanied by growth of standard β‐sheet fractions, indicating a β₂‐to‐β₁ transition as a consequence of the release of solvent from the interstices of peptide assemblies. Our findings highlight the key role played by water molecules in mediating H‐bond formation in β₂‐sheets commonly found in amyloidogenic glutamic acid–rich aggregates.     

Molecular basis of the head‐to‐tail assembly of giant muscle proteins obscurin‐like 1 and titin

Large filament proteins in muscle sarcomeres comprise many immunoglobulin‐like domains that provide a molecular platform for self‐assembly and interactions with heterologous protein partners. We have unravelled the molecular basis for the head‐to‐tail interaction of the carboxyl terminus of titin and the amino‐terminus of obscurin‐like‐1 by X‐ray crystallography. The binary complex is formed by a parallel intermolecular β‐sheet that presents a novel immunoglobulin‐like domain‐mediated assembly mechanism in muscle filament proteins. Complementary binding data show that the assembly is entropy‐driven rather than dominated data by specific polar interactions. The assembly observed leads to a V‐shaped zipper‐like arrangement of the two filament proteins.     

Nanofiber formation of amphiphilic cyclic tri‐β‐peptide

A novel amphiphilic cyclic peptide composed of two β‐glucosamino acids and one trans‐2‐aminocyclohexylcarboxylic acid was synthesized and investigated on assembly formation. The cyclic tri‐β‐peptide was self‐assembled into rodlike crystals or nanofibers depending on preparative conditions. The rodlike crystals showed a layer spacing of 4.8 Å along the long axis, and columnar spacings of 10.8 and 21.5 Å by electron diffraction analysis along the short axis. The former confirms the columnar structure upon molecular stacking, and the latter indicates triple bundle formation of the columnar assemblies. Fourier transform infrared (FT‐IR) measurement of the fibrous assembly showed formation of homogeneous hydrogen bonds among amide groups, also supporting the molecular stacking of cyclic β‐peptides. Straight nanofibers with uniform diameter were also uniquely obtained. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A comparison of convergently evolved insect silks that share β‐sheet molecular structure

Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately β‐sheet with lesser amounts of β‐turns, unordered and α‐helical protein also detected. The orientation of the β‐sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel β‐sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 630–639, 2014.     

Characterization of intrinsically disordered proteins with electrospray ionization mass spectrometry: Conformational heterogeneity of α‐synuclein

Conformational heterogeneity of α‐synuclein was studied with electrospray ionization mass spectrometry by analyzing protein ion charge state distributions, where the extent of multiple charging reflects compactness of the protein conformations in solution. Although α‐synuclein lacks a single well‐defined structure under physiological conditions, it was found to sample four distinct conformational states, ranging from a highly structured one to a random coil. The compact highly structured state of α‐synuclein is present across the entire range of conditions tested (pH ranging from 2.5 to 10, alcohol content from 0% to 60%), but is particularly abundant in acidic solutions. The only other protein state populated in acidic solutions is a partially folded intermediate state lacking stable tertiary structure. Another, more compact intermediate state is induced by significant amounts of ethanol used as a co‐solvent and appears to represent a partially folded conformation with high β‐sheet content. Protein dimerization is observed throughout the entire range of conditions tested, although only acidic solutions favor formation of highly structured dimers of α‐synuclein. These dimers are likely to present the earliest stages in protein aggregation leading to globular oligomers and, subsequently, protofibrils. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Ethanol induced the formation of β‐sheet and amyloid‐like fibrils by surfactant‐like peptide A6K

Self‐assembly of natural or designed peptides into fibrillar structures based on β‐sheet conformation is a ubiquitous and important phenomenon. Recently, organic solvents have been reported to play inductive roles in the process of conformational change and fibrillization of some proteins and peptides. In this study, we report the change of secondary structure and self‐assembling behavior of the surfactant‐like peptide A6K at different ethanol concentrations in water. Circular dichroism indicated that ethanol could induce a gradual conformational change of A6K from unordered secondary structure to β‐sheet depending upon the ethanol concentration. Dynamic light scattering and atomic force microscopy revealed that with an increase of ethanol concentration the nanostructure formed by A6K was transformed from nanosphere/string‐of‐beads to long and smooth fibrils. Furthermore, Congo red staining/binding and thioflavin‐T binding experiments showed that with increased ethanol concentration, the fibrils formed by A6K exhibited stronger amyloid fibril features. These results reveal the ability of ethanol to promote β‐sheet conformation and fibrillization of the surfactant‐like peptide, a fact that may be useful for both designing self‐assembling peptide nanomaterials and clarifying the molecular mechanism behind the formation of amyloid fibrils. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Nanotubular structures developed from whey‐based α‐lactalbumin fractions for food applications

Whey proteins have high nutritional value providing use in dietary purposes and improvement of technological properties in processed foods. Functionality of the whey‐based α‐lactalbumin (α‐La) may be increased when assembled in the form of nanotubes, promising novel potential applications subject to investigation. The purpose of this study was to extract highly pure α‐La from whey protein isolate (WPI) and whey powder (WP) and to construct protein nanotubes from them for industrial applications. For protein fractionation, WPI was directly fed to chromatography, however, WP was first subjected to membrane filtration and the retentate fraction, whey protein concentrate (WPC), was obtained and then used for chromatographic separation. α‐La and, additionally β‐Lg, were purified at the same batches with the purities in the range of 95%–99%. After enzymatic hydrolysis, WPI‐based α‐La produced chain‐like and long nanotubules with ～20 nm width while WPC‐based α‐La produced thinner, miscellaneous, and fibril‐like nanostructures by self‐assembly. Raman and FT‐IR spectroscopies revealed that α‐La fractions, obtained from both sources and the nanostructures, developed using both fractions have some structural differences due to conformation of secondary structure elements. Nanotube formation induced gelation and nanotubular gel network entrapped a colorant uniformly with a transparent appearance. Dairy‐based α‐La protein nanotubules could be served as alternative gelling agents and the carriers of natural colorants in various food processes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1301–1310, 2014     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A hierarchical order within protein structures underlies large separations between strands in β‐sheets

Protein β‐sheets often involve nonlocal interactions between parts of the polypeptide chain that are separated by hundreds of residues, raising the question of how these nonlocal contacts form. A recent study of the smallest β‐sheets found that their formation was not driven by signals hidden in the primary sequence. Instead, the strands in these sheets were either local in sequence, or, when separated by large sequential distances, the intervening residues were found to fold into compact modules that anchored distant parts of the chain in close spatial proximity. Here, we examine larger β‐sheets to investigate the extensibility of this principle. From an analysis of the β‐sheets in a nonredundant protein dataset, we find that a highly ordered hierarchical relationship exists in the intervening structure between nonlocal β‐strands. This observation is almost universal: virtually all β‐sheets, no matter their complexity, appear to adopt an antiparallel model to manage the nonlocal aspects of their assembly, one where the chain, having left the vicinity of an unfinished β‐sheet, retraces its steps via the same route to complete the initial sheet. Exceptions typically involve unstructured regions at chain termini. Moreover, an analysis of the residues involved in nonlocal crossstrand interactions did not produce any evidence of a signal hidden in the sequence that might direct long‐range interactions. These results build on those reported for the smallest sheets, suggesting that sheet formation is either local in sequence or local in space following prior folding events that anchor disparate parts of the chain in close proximity. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Structural basis of intramitochondrial phosphatidic acid transport mediated by Ups1‐Mdm35 complex

Ups1 forms a complex with Mdm35 and is critical for the transport of phosphatidic acid (PA) from the mitochondrial outer membrane to the inner membrane. We report the crystal structure of the Ups1‐Mdm35‐PA complex and the functional characterization of Ups1‐Mdm35 in PA binding and transfer. Ups1 features a barrel‐like structure consisting of an antiparallel β‐sheet and three α‐helices. Mdm35 adopts a three‐helical clamp‐like structure to wrap around Ups1 to form a stable complex. The β‐sheet and α‐helices of Ups1 form a long tunnel‐like pocket to accommodate the substrate PA, and a short helix α2 acts as a lid to cover the pocket. The hydrophobic residues lining the pocket and helix α2 are critical for PA binding and transfer. In addition, a hydrophilic patch on the surface of Ups1 near the PA phosphate‐binding site also plays an important role in the function of Ups1‐Mdm35. Our study reveals the molecular basis of the function of Ups1‐Mdm35 and sheds new light on the mechanism of intramitochondrial phospholipid transport by the MSF1/PRELI family proteins.     

High resolution crystal structure of Clostridium propionicum β‐alanyl‐CoA:ammonia lyase,a new member of the “hot dog fold” protein superfamily

Clostridium propionicum is the only organism known to ferment β‐alanine, a constituent of coenzyme A (CoA) and the phosphopantetheinyl prosthetic group of holo‐acyl carrier protein. The first step in the fermentation is a CoA‐transfer to β‐alanine. Subsequently, the resulting β‐alanyl‐CoA is deaminated by the enzyme β‐alanyl‐CoA:ammonia lyase (Acl) to reversibly form ammonia and acrylyl‐CoA. We have determined the crystal structure of Acl in its apo‐form at a resolution of 0.97 Å as well as in complex with CoA at a resolution of 1.59 Å. The structures reveal that the enyzme belongs to a superfamily of proteins exhibiting a so called “hot dog fold” which is characterized by a five‐stranded antiparallel β‐sheet with a long α‐helix packed against it. The functional unit of all “hot dog fold” proteins is a homodimer containing two equivalent substrate binding sites which are established by the dimer interface. In the case of Acl, three functional dimers combine to a homohexamer strongly resembling the homohexamer formed by YciA‐like acyl‐CoA thioesterases. Here, we propose an enzymatic mechanism based on the crystal structure of the Acl·CoA complex and molecular docking. Proteins 2014; 82:2041–2053. © 2014 Wiley Periodicals, Inc.     

The rod domain of NF-L determines neurofilament architecture, whereas the end domains specify filament assembly and network formation

Neurofilaments, assembled from NF-L, NF-M, and NF-H subunits, are the most abundant structural elements in myelinated axons. Although all three subunits contain a central, alpha-helical rod domain thought to mediate filament assembly, only NF-L self-assembles into 10-nm filaments in vitro. To explore the roles of the central rod, the NH2- terminal head and the COOH-terminal tail domain in filament assembly, full-length, headless, tailless, and rod only fragments of mouse NF-L were expressed in bacteria, purified, and their structure and assembly properties examined by conventional and scanning transmission electron microscopy (TEM and STEM). These experiments revealed that in vitro assembly of NF-L into bona fide 10-nm filaments requires both end domains: whereas the NH2-terminal head domain promotes lateral association of protofilaments into protofibrils and ultimately 10-nm filaments, the COOH-terminal tail domain controls lateral assembly of protofilaments so that it terminates at the 10-nm filament level. Hence, the two end domains of NF-L have antagonistic effects on the lateral association of protofilaments into higher-order structures, with the effect of the COOH-terminal tail domain being dominant over that of the NH2-terminal head domain. Consideration of the 21-nm axial beading commonly observed with 10-nm filaments, the approximate 21-nm axial periodicity measured on paracrystals, and recent cross-linking data combine to support a molecular model for intermediate filament architecture in which the 44-46-nm long dimer rods overlap by 1-3-nm head-to-tail, whereas laterally they align antiparallel both unstaggered and approximately half-staggered.     

Chaperone‐like activities of different molecular forms of β‐casein. Importance of polarity of N‐terminal hydrophilic domain

As a member of intrinsically unstructured protein family, β‐casein (β‐CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone‐like activity in vitro. Like many chaperones, native β‐CN does not contain cysteinyl residues and exhibits strong tendencies for self‐association. The chaperone‐like activities of three recombinant β‐CNs wild type (WT) β‐CN, C4 β‐CN (with cysteinyl residue in position 4) and C208 β‐CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native β‐CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (β‐CND) of C4‐β‐CN and C208 β‐CN were also studied and their chaperone‐like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT β‐CN, C208 β‐CN, C4 β‐CN and C4 β‐CND exhibited significantly lower chaperone‐like activities than native β‐CN. Dimerization of C208 β‐CN with two distal hydrophilic domains considerably improved its chaperone‐like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N‐terminal hydrophilic domain as important functional elements in enhancing the chaperone‐like activity of native β‐CN. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 623–632, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

An alternative structural isoform in amyloid‐like aggregates formed from thermally denatured human γD‐crystallin

The eye lens protein γD‐crystallin contributes to cataract formation in the lens. In vitro experiments show that γD‐crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV‐B photodamage results in its C‐terminal domain forming the β‐sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet‐like aggregates that contain cross‐linked oligomers of the protein, according to transmission electron microscopy and SDS‐PAGE. We use two‐dimensional infrared spectroscopy to show that these aggregates have an amyloid‐like secondary structure with extended β‐sheets, and use isotope dilution experiments to show that each protein contributes approximately one β‐strand to each β‐sheet in the aggregates. Using segmental ¹³C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N‐terminal and C‐terminal domains contribute to β‐sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N‐terminal domain into the fiber structure to intermolecular cross linking.     

Conformation change of hornet silk proteins in the solid phase in response to external stimulation

Hornet silks adopt a variety of morphology such as fibers, sponge, films, and gels depending on the preparation methods. We have studied the conformation change of hornet silk proteins (Vespa mandarina) as regenerated films, using chiroptical spectrophotometer universal chiroptical spectrophotometer 1, which can measure true circular dichroism spectra without artifact signals that are intrinsic to solid‐state samples. The spectra showed that the proteins in films alter the conformation rapidly from the α‐helix to the coiled coil and then to a β‐sheet structure in response to heat/moisture treatment, but the transformation stopped at the coiled coil state when the sample was soaked in EtOH/water solution. Water is required for the α‐helix to the coiled coil transition, and extra energy is required for the further transition to a β‐sheet structure. This is the first successful circular dichroism study of fibril silk proteins to follow the conformation changes in the solid state. This work shows that proteins can undergo conformational changes easily even in the solid phase in response to external stimuli, and this can be traced by solid‐phase‐feasible chiroptical spectrophotometers. Application of unnatural stress to proteins gives valuable insights into their structure and characteristics.