Transformation of Coiled Î±-Helices into Cross-Î²-Sheets

Subscriber access provided by READING UNIV

Article

Transformation of Coiled #-Helices into cross-#-Sheets Superstructure Taiyo Yoshioka, Tsunenori Kameda, Kohji Tashiro, Noboru Ohta, and Andreas K. Schaper Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00920 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 2, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Transformation of Coiled α-Helices into cross-β-Sheets Superstructure

Taiyo YOSHIOKA1, Tsunenori KAMEDA1*, Kohji TASHIRO2, Noboru OHTA3, Andreas K. SCHAPER4* 1

Silk Materials Research Unit, National Agriculture and Food Research Organization (NARO), 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan

2

Department of Future Industry-oriented Basic Science and Materials, Graduate School of Engineering, Toyota Technological Institute, Tempaku, Nagoya 468-8511, Japan

3

Japan Synchrotron Radiation Research Institute, 1-1 Koto, Mikazuki-cho, Sayo-gun, Hyogo 679-5198, Japan 4

Center for Materials Sciences, Philipps University of Marburg, 35032 Marburg, Germany

Hornet silk, Coiled-coil, cross-β-sheet, Helix-to-sheet transition, Synchrotron X-ray analysis 1

ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The fibrous silk produced by bees, wasps, ants or hornets, is known to form a four-strand αhelical coiled coil superstructure. We have succeeded in showing the formation of this coiled coil structure not only in natural fibers but also in artificial films made of regenerated silk of the hornet Vespa simillima xanthoptera using wide- and small-angle X-ray scatterings and polarized Fourier transform infrared spectroscopy. On the basis of time-resolved simultaneous synchrotron X-ray scattering observations for in situ monitoring of the structural changes in regenerated silk material during tensile deformation, we have shown that the application of tensile force under appropriate conditions induces a transition from the coiled α-helies to a cross-β-sheet superstructure. The four-stranded tertiary superstructure remains unchanged during this process. It has also been shown that the amorphous protein chains in the regenerated silk material are transformed into conventional β-sheet arrangements with varying orientation.

1.

INTRODUCTION

The silk of the Lepidopterans, including the domestic silkworm (Bombyx mori), shows highly repetitive sequences in the amino acids of the constituent peptide chains preferentially adopting a repeated β-turn type II (silk I) conformation during fibroin synthesis in the silk gland where it is stabilized under distinctive physiological conditions.1 The silk feedstock is subjected to shear and extensional flow in the fiber spinning process which initiate its conversion into a crystalline β-sheet structure (silk II)2 (see Figure 1). Similarly, the dominant regions in the high-strength silk structure of spiders (Araneae) are oriented spidroin β-sheets formed out of 31-helical or α-helical conformation chains or of random coils present in the glands.3-5 In contrast, the silk proteins of aculeates (stinging Hymenopterans) are characterized by quite different features in their amino acid sequences.6 Each silk protein contains alanine-rich domains with a repetition of a heptad sequence motif (a-b-c-d-e-f-g)n, in 2


Page 2 of 43

Page 3 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

which the positions a and d contain hydrophobic residues and the other positions (b, c, e, f and g) are hydrophilic with a high probability. Such heptad amino acid sequences tend to form a coiled coil structure, in which multiple α-helices are mutually entwined.7-9 The properties of these silk fibers are expected to differ considerably from both silkworm and spider silks. The yellow hornet Vespa simillima xanthoptera Cameron, investigated in this study, is one of the most representative hornets in Japan and produces nests of over one meter in diameter in large case. The silk was found to comprise four major proteins, whose amino acid sequences have been completely identified by the Kameda group10 (see Section 3.5 for further details). The revealed amino acid sequences predict the preferential formation of a coiled coil structure, as was found by Atkins11 in his early work with silk of the honeybee. Our recent analyses using X-ray and electron diffraction have indeed provided evidence of the fourstranded supercoil structure present in hornet silk.12 Kameda et al.13 also developed techniques to regenerate the silk in aqueous solution. Moreover, they could successfully fabricate transparent and drawn gel films14 with tensile strengths of up to 170 MPa, and a modulus of 5.5 GPa, higher than those of films prepared from regenerated silkworm silk.15 The dominant folding regime was found to change from coiled coil α-helices to β-sheet conformations with mechanical stretching.14 In association with the conventional β-sheet structure, here we call it parallel-β-sheet without distingushing the parallelism of molecular chain stems (parallel or anti-parallel types), we notice the presence of another type of the β-structure, which is called the cross-βstructure (see Figure 1). The cross-β-sheet structure is known as the particular folding principle in the silk of special insects16-19 and bacterial flagella,20 and was also found in fibrillar entities in the spider gland.21 It is suggested that parts of the cross-β-phase are also initially linked to the terminal domains of nonrepetitive segments in the protein chain,22 but 3


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

are capable of triggering the α - β transformation in the repetitive parts.3 The cross-β-sheet in a stretched fiber of recombinant honeybee silk was found to coexist with the α-helical coiled coil.23 In another context, the cross-β-conformation has gained tremendous attention as an evolutionary highly conserved structure of protein misfolding in living systems, particularly in the amyloid fibrilogenesis.24 These protein mutations are known to cause a number of fatal diseases such as Alzheimer, Parkinson, and Huntington, as well as several clinical syndromes caused by pathogenic prions.25,26 Intriguingly, the amyloids are ambivalent in their functioning as they also fulfill positive tasks in the organism.27 Studies on the denaturation of proteins by non-physiological organic solvents, inappropriate pH levels, elevated temperature or other forms of stress, including mechanical deformation and distortion, are key in understanding of the general principles of amyloidogenesis. However, the direct observation of the formation of the cross-β-structure based on the denaturation of proteins has not been realized in any silk material. On the basis of Fourier transform infrared (FTIR) spectroscopy, Litvinov et al. pointed out the possibility of the involvement of the cross-β-structure in the transformation from the α-helical coiled coil to the β-sheet structure in the stretching of viscoelastic hydrated fibrin clots.28 In this study, we have successfully obtained experimental evidence of the transformation of α-helical coiled coils into cross-β-sheets that occurred during the stretching of the hornet silk gel film of Vespa simillima xanthoptera using wideand small-angle X-ray scattering (WAXD and SAXS), polarized FTIR spectroscopy and timeresolved synchrotron X-ray analysis. The obtained knowledge is believed to be quite important in understanding the principles of amyloidogenesis.

2.

EXPERIMENTAL SECTION

2.1.

Materials 4


Page 4 of 43

Page 5 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Short filaments of approximately 5 mm in length were collected from the nest combs of the hornet Vespa simillima xanthoptera Cameron, and carefully bundled together in parallel. Regenerated hornet silk gel films were prepared according to the procedure reported previously.10,13 Briefly, the cocoon caps were dissolved in 9M LiBr aqueous solution by stirring for 15 min at 37 °C, and then dialyzed at 20 °C for 4 days in sterilized water using a cellulose tube (cut-off molecular weight 18000; Wako Pure Chemical Industries Ltd., Japan). During the dialysis the solution gradually transformed into a turbid gel. The obtained hydrogel was then sandwiched between water-absorbing papers, whichｈ were then subjected to pressure by applying a force of 35 N for three days. Finally, the cellulose tube was removed and the films were completely dried at free pressure in vacuum for 24 h at room temperature (~20 °C) to obtain the hornet silk gel film. The resultant film was approximately 0.25 mm in thickness.

2.2.

Characterization of the native hornet silk and gel films from regenerated silk

2.2.1. WAXD studies The crystalline modification and orientation of both the native hornet silk fiber and the silk gel film were investigated by WAXD analysis with an R-Axis Rapid II X-ray diffractometer (50 kV, 100 mA, Mo-Kα) (Rigaku Co., Japan) equipped with a cylindrical imaging plate camera with a pixel size of 100 × 100 µm2. Cerium oxide was used to calibrate the camera distance. A free software FIT2D (developed by A. P. Hammersley of European Synchrotron Radiation Facility, Grenoble) was used for subtracting the air-scattering and for extracting the q-, 2θ-, and azimuthal-profiles from the 2-dimensional WAXD and SAXS patterns.

2.2.2. SAXS studies 5


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The superstructures of native hornet silk fiber, regenerated gel film, and hot-stretched gel films were investigated by SAXS analysis. The experiment was performed at SPring-8 beam line 40B2 (Hyogo, Japan). The wavelength of an incident X-ray beam was 0.0709 nm. The beam size at the sample position was roughly 600 µm in diameter. The sample-to-camera distance was 2000 mm. Silver behenate was used to calibrate the camera distance. An imaging plate system of R-Axis VII (Rigaku Co., Japan) with a pixel size of 100 × 100 µm2 was used as a detector.

2.2.3. Polarized ATR- investigations Seven pieces rectangular strips (30 mm in length × 3 mm in width) were cut out of the original gel-film with 0.25 mm thickness. Each strip was hot-stretched in an oil-bath at 110 °C at various draw ratios from 30 to 120 % strain. The structural study of the hot-stretched gel-films and the original un-stretched one was performed by polarized attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy using an FTS 7000 FTIR spectrophotometer (Varian, USA) equipped with an ATR accessory (Miracle, Pike Technologies, Inc., USA). A diamond crystal was used as an internal optical element for the ATR measurement. The wire-grid polarizer was set in the optical pass of the ATR cell. Recording of the ATR-FTIR spectra shown in this manuscript was repeated until the reproducibility of each spectrum was guaranteed.

2.2.4. Time-resolved synchrotron X-ray scattering investigations The structural change of the regenerated hornet silk gel film occurring in the wet-stretching process was investigated by the time-resolved simultaneous measurement of the stress-strain curve and synchrotron X-ray scattering. The experiment was performed at the SPring-8 beam 6


Page 6 of 43

Page 7 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

line 40B2 (Hyogo, Japan) (see Figure 2). The wavelength of the irradiant X-ray beam (λ) was 0.0709 nm. The beam size at the sample position was roughly 600 µm in diameter. The sample-to-camera distance was 390 mm, and an R-Axis VII imaging plate system of (Rigaku Co., Japan) with a pixel size of 100 × 100 µm2 was used as a detector. The scattering vector q (defined as 4π·sinθ /λ) covered the range from 1 to 25 nm-1. The regenerated hornet silk gel film was cut into rectangular samples with dimensions; 60 mm length × 3.2 mm width × 0.37 mm thickness. A central region of 20 mm length was immersed in water for 30 min before the stretching experiment started. After a quick transfer to the stretching device (micro-stretcher, Linkam Scientific Instruments Ltd., UK) equipped with a handmade humidity chamber (Figure 2), stretching was performed at a rate of 1.2 mm/min. The relative humidity in the humidity chamber was maintained over RH 95%. The time-resolved X-ray scattering measurement during stretching was carried out at time intervals of 80 s, each roughly corresponding to a strain of 8%. The incident X-ray beam direction was along the normal to the gel film (through-direction).

3.

RESULTS AND DISCUSSION

To achieve a comprehensive picture of the structure and mechanisms of the tension-induced transitions of regenerated gel films of silk from the hornet Vespa simillima xanthoptera, we describe in Section 3.1 our observations of the coiled coil structure of native fibers of hornet silk and compare them with those reported previously of the fibers from Vespa mandarinia japonica.12 In Section 3.2, we report structural investigations on regenerated silk gel films and discuss differences in the coiled coil conformations compared with the ones from native fibers. The focus of Section 3.3 is the elucidation of two different types of β-sheet structure as revealed in the stretched gel films. In Section 3.4 an attempt is made to clarify the basis of the 7


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

time-resolved synchrotron X-ray scattering measurements, the mechanisms of the transformation of the coiled α-helix into the cross-β-sheet ordered structure during in situ stretching, keeping the four-strand superstructure unchanged. Finally, in Section 3.5 we are trying to associate each structural formation with a specific character of amino acid sequence in the hornet silk. Further, we give an inclusive hierarchical model for the tension induced structural transformation observed in the regenerated hornet silk.

3.1.

Structure of the native hornet silk fiber

Previous observations made using of electron diffraction revealed a four-strand coiled coil structure of native silk from Vespa mandarinia japonica. The axial periodicity of the coiled coil was determined using a laboratory X-ray diffractometer to be approximately 344 Å.12 Figure 3 presents new measurements of native fibers from Vespa simillima xanthoptera using two-dimensional synchrotron SAXS. Synchrotron radiation brought a dramatically improved resolution, a much higher signal-to-noise ratio, and a larger covered q-range compared to conventional SAXS measurements. In Figure 3b, 21 peaks from the 3rd to the 25th order of layer scattering are successfully detected within in a periodicity of 343.3 ± 0.2 Å. Repeated measurements shown in S.I.1 of Vespa mandarinia japonica fibers using synchrotron SAXS have revealed 20 peaks from the 4th to the 25th layer scattering in agreement with a periodicity of 342.7 ± 0.3 Å. The characteristics of a coiled coil structure in the native fiber of Vespa simillima xanthoptera is confirmed also by WAXD analysis. A two-dimensional WAXD pattern is shown in Figure 4a and the corresponding diffraction profiles along the equatorial and meridional lines are shown in Figures 4b and c. The dominant diffraction features of coiled coils can clearly be deduced from the data: a strong meridional peak at ~5.1 Å related to the helical pitch of a supercoiled α-helix tilted against the fiber direction by ~19°. According to 8


Page 8 of 43

Page 9 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

the fundamental analysis by Crick7, Pauling and Corey8 and Atkins11 the pitch of the straight

α-helix of ~5.4 Å reduces to ~5.1 Å, if four α-helices are entwined into a coiled coil superhelix (cos-1 5.1/5.4 = 19o). The strong and broad equatorial scattering peak with a maximum at 8.6 Å in Figure 4c represents the mean distance between adjacent α-helices within the coiled coil.29,30 A closer look at the meridional scattering tells us that the strong ~5.1 Å meridional peak is composed of two peaks: one sharp peak at 5.1 Å and one broader peak at 5.0 Å (see the inset in Figure 4b). The latter value is equivalent to a tilting angle of ~22°. Following the analysis of side-chain configurations in double-stranded hard keratin by Busson et al.,31 deviations like this may be attributed to molecular distortions such as helical disorder (see Section 3.2).

3.2.

Structure of the as-prepared hornet silk gel film

The two kinds of WAXD pattern of the as-prepared unstretched gel film, obtained by irradiating the object with the X-ray beam along the mutually perpendicular edge and through-direction, are shown in Figures 4d and f, respectively. Whereas in the through-view the WAXD pattern shows powder ring reflections, the edge-view contains clear signs of orientation. The q-profile of the through-view pattern is shown in Figure 4e, and the equatorial and meridional q-profiles of the edge-view pattern are given in Figures 4g and h. The combined observation of a meridional arc at ~5.0 Å and strong equatorial scattering at ~8.6 Å means that the helical axes of the coiled-coils are oriented within the plane of the film. This significant in-plane orientation is suggested to be caused by the high compression stress applied in the gel film fabrication process.

9


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In contrast to the case of native silk fiber, where the sharp 5.1 Å and a broader 5.0 Å peak were clearly distinguishable (Figure 4b), the latter was dominant in the as-prepared gel film (see insets in Figures 4e and g). This suggests that the majority of the coiled coil structure formed in the gel film is not perfectly coiled in a regular manner, but somewhat distorted causing a larger tilting angle of the α-helices (~22°) compared with that at perfect packing (~19°). The broadness of the peak indicates a distribution of the tilting irregularity along the α-helical segments. Further evidence of a coiled coil protein superstructure in the unstretched gel film was confirmed by two-dimensional through- and edge-view SAXS patterns in Figures 5a and b. The meridional and equatorial line profiles of the edge-view pattern are shown in Figures 5c and d, respectively. The strong in-plane orientation, which was already deduced from the anisotropic WAXD patterns, are supported by SAXS. Many sharp peaks are detected along the meridional direction in the edge-view pattern from which an axial periodicity of ~364 Å can be estimated. This value is slightly larger than the ~343 Å found in the native fibers of Vespa simillima xanthoptera silk (see Figure 3) or the ~344 Å of the silk from Vespa mandarinia japonica.12 In the equatorial profile of the edge-view pattern, two strong scattering peaks giving a periodicity of 36.2 Å are detected. This periodicity is suggested to correspond to a layer pitch of the plane-oriented coiled coil structures, as schematically depicted in Figure 6. Based on the X-ray diffraction patterns of honeybee silk, Fraser and Parry32 recently proposed a packing model in a supercell of ropes built up by four four-strand coiled coils with a two-dimensional lattice of a = 45.2 Å, b = 37.4 Å and γ = 101.1°. The 36.2 Å spacing detected in the four-strand coiled coil structure of the hornet silk gel films is in close agreement with a unit cell of similar size.

10


Page 10 of 43

Page 11 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

A significant difference between the WAXD patterns of the native fiber and the asprepared gel film has to be noted: The detection of a strong 4.6 Å peak is observed only in the gel film (Figure 4). This scattering produces a ring pattern in the through-view, but a meridional scattering arc in the edge-view (Figures 4f and g). It can be assigned to the 200 lattice spacing of β-sheet crystallites, with the a-axis of the β-sheets (or the direction of intermolecular hydrogen bondings33) preferentially oriented within the film plane.

3.3.

Structure of the regenerated hornet silk gel film after hot-stretching The prepared gel film can be stretched easily under

3.3.1. X-ray scatatering data

conditions of elevated temperature or wetting up to a maximum strain of approximately 120% (up to a draw ratio of ~2.2). Further stretching leads to film rupture under both wet and hot conditions. Figure 7a shows WAXD patterns taken of three different views in the directions through, edge and end, of the gel film hot-stretched by 120% strain at 110 °C [The WAXD pattern for the stretched sample shown in Figure 5a rightside is the same as that of the edge direction given in Figure 7a]. The equatorial and meridional 2θ-profiles (θ = scattering angle) scanned from the edge-view WAXD pattern are shown in Figures 6b. Some characteristic structural changes can be observed when compared with the original unstretched film (Figure 4). First, the reflection related to the helical pitch of coiled α-helices (~5.0 Å), which was detected for the unstretched film, disappeared. This means the coiled coil structure has become destabilized by stretching. This is confirmed by SAXS measurements (Figure 5c). In the edge-view SAXS profile of the hot-stretched gel film no specific scattering from the coiled coil superlattice was detected. The second important point is about the intensity distribution of the diffraction peak of the 200 β-sheet structure (β200) observed in the through-view pattern. The distribution was 11


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

almost homogeneous before stretching (see Figure 4d), while the ring pattern was separated into two different arcs on the meridian and the equator after stretching, as shown in Figure 7a. As known from the azimuthal profiles of the β200 reflections (Figure 7c), the intensity maximum is located on the equatorial direction and also on the near-meridional line with ~19° deviation from the meridional direction. This is recognized in both the through- and edgeview patterns. Since the plane orientation makes the detailed analysis of the WAXD pattern complicated, fully-rotated WAXD patterns (left side in Figure 7d) were obtained by rotating the hot-stretched gel-film around the stretching axis. The separation of 200 reflection into two directions is clear. At this moment we have reached a quite important finding: the β200 reflection arcs around the meridional line (shown by yellow color in right side in Figure 7d) are caused by a cross-β-sheet structure, in which the a-axis is oriented by ~19° against the direction perpendicular to the stretching axis (refer to Section 3.4.1). The angle 19° is almost the same as the angle between the α-helical axis and the coiled coil axis. In contrast, the other 200 reflection detected along the equatorial line (blue line) belongs to a parallel-β-sheet structure with the chain direction oriented parallel to the stretching direction. The X-ray diffraction patterns of the stretched gel film can thus be interpreted by assuming the coexistence of parallel-β- and cross-β-sheet structures. The two-dimensional X-ray diffraction patterns were simulated for the parallel-β- and cross-β-structures as shown in Figure 8. The parallel-β-structure model used in Figure 8a was that proposed by Takahashi et al.33 (refer to Figure 1; the unit cell parameters are a = 9.38 Å, b = 9.49 Å, and c (fiber axis) = 6.98 Å). The cross-β-structure model (shown in Figure 1) was created by minimizing the Takahashi model with the chain folding, where the energy minimization was performed using a COMPASS force field.34 The thus-obtained cross-β model shows the intramolecular hydrogen bonds between the chain stems with the short 12


Page 12 of 43

Page 13 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

segmental lengths. The X-ray diffraction patterns were calculated using a commercial software Cerius2 (Accelres, Biovia, version 4.10), where the wavelength of the incident X-ray beam was 0.71 Å, the isotropic temperature factor 5 Å2, the crystallite size 200 × 200 × 200 Å3, and the degree of orientation 5° (half-width). Figure 8b shows the X-ray diffraction pattern of the cross-β-sheet structure. The strong 200 reflection is detected with the angle about 13o from the meridional direction, similar to the observed data (Figure 8c). The combination of the X-ray diffraction patterns of the cross-β- and parallel-β-forms gives the pattern shown in Figure 8d, which corresponds well to the X-ray diffraction pattern obtained for the sample stretched about 100%. Further accurate and quantitative comparisons need to be done by analyzing the 2-dimensional X-ray diffraction data of the pure and highly-oriented cross-β sample. These simulations allow us to speculate the transition process from the bundle of α-helices to the aggregated cross-β-sheet structure in the following way. The application of a shear stress to the α-helix is speculated to cause the deformation of the helix and generate the short planar-zigzag-type segments combined with the intramolecular hydrogen bonds. These cross-β-sheets are generated in the neighboring coiled coils of αhelical chains cooperatively and are stacked together to form the aggregation state of the cross-β chain segments to give the X-ray diffraciton spots.

3.3.2. IR spectra

To getting additional information we have analyzed the vibrational

FTIR spectral data. The samples were gel films hot-stretched at various draw ratios of ε = ~30 to 100 %. The geometric relationship between the reflection plane, the direction of the electric field vector of an incident polarized-IR beam and the stretch direction of the gel-film is schematically shown in Figure 9a. The stretching direction (S.D.) is perpendicular to the reflection plane. Polarized ATR-FTIR spectra measured for the unstretched and hot-stretched 13


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

gel films are shown in Figure 9b, where the bands of the amide-I (C=O stretching mode) and amide-II (CN stretching mode) are reproduced. The transition dipole vector of the amide-I band is nearly parallel to the α-helical axis and that of the β-sheet structure is perpendicular to the zigzag chain axis. Oppositely, for the amide-II band, the C-N transition dipole is nearly perpendicular to the α-helical axis and parallel to the β-sheet zigzag chain axis. The polarized spectra, measured using polarized IR beams with E// and E⊥ electric vectors, together with the unpolarized spectra are shown in green, red and blue colours in Figure 9. The polarized IR spectra of the undrawn film consists of α-helix and β-sheet bands. Stretching of the film caused the α-helix bands to disappear whereas the β-sheet bands increase in intensity, indicating a structural transition from the α-helix to β-sheet structure. This is consistent with the above-mentioned X-ray results. It should be noted that the amide-I band of the β-sheet detected for the sample stretched by 100% consists of the two components: the 1625 cm-1 and 1617 cm-1 bands. Further, these bands show different polarization character. As mentioned above, our experimental setting satisfies the following relations: cross-β → (C=O dipole // stretchign direction) → (zigzag chain ⊥ stretching direction) prallel-β → (C=O dipole ⊥ stretching direction) → (zigzag chain // stretching direction) Thus, the “parallel red” band at 1617 cm-1 in the polarized ATR spectra measured under the polarization character of C=O dipole directed along the stretching direction should correspond to cross-β-sheet. On the contrary, the “perpendicular green” band at 1625 cm-1 measured under the polarization character of C=O dipole directed perpendicular to the stretching direction must correspond to parallel-β-sheet. In this way, the polarized ATR spectral data demonstrated that the two components of the β-sheet structure exist in the stretched gel film as suggested by X-ray diffraction analysis. The difference in the peak 14


Page 14 of 43

Page 15 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

position of the vibrational frequencies between these two bands indicates that the paralleland cross-β-sheets are not simply different in the orientation direction with respect to the drawing axis but originates from the difference in the structural situation. As shown in Figure 1, the cross-β is built by the parallel arrays of the intramolecularly-hydrogen-bonded short zigzag segments with the folding structure, while the parallel-β consists of the parallel arrays of the long zigzag segments joined together by the intermolecular-hydrogen-bonds. In the literature, these two structures are called amyloid-like and normal β-sheet structure, respectively, and the structural difference might result in the difference in the IR band frequencies.35,36 These assignments are also confirmed from the changes of band intensities depending on the stretching ratio. As the sample was stretched, the band component at 1617 cm-1 increased in intensity (strain ε = 30% ~ 60% ) and decreased (100%), while the band at 1625 cm-1 became stronger finally at ε = 100%. Figure 9c shows the strain dependence of the β-sheet bands, where the absorbance (A) of the perpendicularly-polarized 1617 cm-1 band was divided by the absorbance of the perpendcularly-polarized 1515 cm-1 band, i.e., A(⊥)

-1 (1617cm )

/A(⊥)

-1 (1515cm ).

Taking into

account the same behavior of the parallel-β-band at 1625 cm-1, the band at 1515 cm-1 may be assigned to the parallel-β-sheet. Therefore, the ratio of absorbance between the 1617 and 1515 cm-1 bands corresponds to the ratio of relative amount between the cross-β and parallel-

β structures (More ideally, these two band intensities can be plotted separately against the strain, but their ratio had to be used here in order to erase the effect of the variation of penetration depth of the incident IR beam in the ATR measurement due to the different sample surface conditions). The relative intensity of the 1617 cm-1 band started to increase significantly up to a drawing point of ~50%. The behavior of the 1617 cm-1 band is consistent with the X-ray observation (see Figure 10), suggesting the assignment of this band to the 15


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cross-β-form: the cross-β-form increases in amount in the strain region up to 50% and then decreases, and the parallel-β-sheet component increases beyond this critial strain. Here, one comment is added about the reason why the starting gel film used for the ATR spectral measurement contains the cross-β-sheet in addition to the dominantly-existing

α-coiled coils. As mentioned in Section. 2.1.2, in the preparation of the gel film a compression force of 35 N was applied to the silk hydrogel. A comparison of the ATR-FTIR amide-I bands of gel films prepared under free force and those by applying a high compressive force (S.I.2) clearly reveals a higher intensity of the cross-β-sheet bands in the compressed gel than in the uncompressed gel. These results show that the application of pressure boosts the structural transformation of parts of α-helices into cross-β-sheets during the drying process of the regenerated hydrogel.

3.4. Structural transformation of the gel film during wet-stretching 3.4.1. Transition from the coiled α-helix into the cross-β-sheet conformation The unoriented hornet silk gel film has so far been proved to be composed of two kinds of crystal structures, the coiled coil structure (coiled α-helices) and the β-structure of the cross-

β-type both in strong in-plane orientation. We observed that the coiled coil structure disappeared at ~100% strain of hot-stretching, and the dominant structure changed into the coexistence of the cross-β- and parallel-β-sheets. To investigate the details of the structural transition, simultaneous measurements were performed for the stress-strain curve and the time-resolved synchrotron WAXD pattern during the stretching deformation process of the hornet silk gel film. Because of technical aspects of the experimental setup, wet-stretching was adopted for this purpose. As will be shown below, the structural transition phenomenon was confirmed to be essentially the same for the hot- and wet-stretching methods. 16


Page 16 of 43

Page 17 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

The stress-strain curve of wet-stretching is shown in Figure 10e. The time-resolved changes in the WAXD patterns during wet-stretching are shown in Figure 10a and a schematic drawing is shown in Figure 10b. The crystalline diffraction rings characteristic of the coiled α-helix and β-sheet structures are detected in the initial unstretched gel film (0% strain in Figure 10b). The diffraction pattern changed into an oriented pattern upon drawing: The diffraction ring corresponding to the coiled α-helix pitch indicated as Cα in Figure 10 changed gradually into arcs with the intensity maximum concentrated nearly on the meridian (see the pattern at 47% strain). With increasing stretching ratio the β200 reflection continuously decreased in angular width. At even higher stretching ratio, the β200 diffraction spot appeared on the equator. The sharp reflection of the coiled α-helix had nearly disappeared at 118% strain as shown in Figure 10b. The q-profiles of the reflection peaks 5.0 and 5.1 Å scanned for the whole azimuthal angle are shown in Figure 10c. In the dry state, the 5.1 Å meridional peak was not observed distinctively for the unstretched gel film, but appeared clearly as in the native dry fiber after wetting (see top profile in Figure 10c). The change of the amont of coiled α-helices was evaluated from the change of peak intensity in the sharp peak at 5.1 Å as plotted in Figure 10e. For the estimation of the change of amounts of cross-β- and parallel-β-sheets, the azimuthal profiles of the β-sheet reflection at 4.6 Å (cross- (×-) β200 and parallel- (//-) β200) are evaluated (see Figure 10d). The peak separation was performed for these azimuthal profiles in the same manner used in Figure 7c and the change of peak intensities of the parallel- and cross-β-sheets were also plotted in Figure 10e. On the other hand, to evaluate the orientation changes of the originally formed coiled α-helices and β-sheets, the q -profiles were obtained by meridional and equatorial scans in the limitted ±10° azimuthal range (see

17


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

S.I.3a and b). The peak intensity changes of the coiled α-helix and the β-sheets were evaluated as shown in S.I.3c. Although the total amount of coiled α-helix material did not change in the starting 20% strain region (i) in Figure 10e, the intensity of the corresponding meridional reflection increased (S.I.3c). This indicates that the c-axis orientation of the coiled coils along the stretching direction increased in this strain region. In the same strain region, the β200 reflection intensity along the equatorial line decreased and the β200 reflection component along the meridional line increased (see S.I.3c). This is consistent with the conclusion reached from the polarized ATR-FTIR analysis. The intensity of the coiled α-helix peak at 5.1 Å had started to decrease and the 4.6 Å peak reflection of the cross-β-sheets had started to increase at the same timing at a strain of approximately 20% (region (ii) in Figure 10e). These are strong signs of a structural transition from the coiled α-helices to cross-β-sheets. Furthermore, the initiation of the plateau region in the stress-strain curve at the same time also gives evidence of this structural transition as it is known from similar phenomena during the crystal transformation in regenerated silk of Bombyx mori,37 and in synthetic materials such as poly(tetra-methylene terephthalate)38 and poly(ethylene oxide).39 On the basis of these experimental data, we conclude that, in fact, a tension-induced structural-transition from the coiled α-helices into cross-β-sheets has occurred. Here we focus on the morphology of the thus-transformed cross-β-sheets. The experimental data necessary for the discussion may be summarized as follows. (i) The X-ray 002 reflection of the β-sheet structure is observed at about 19° from the drawing direction, which is close to the angle of the α-helical axis measured from the coiled coil axis. (ii) The equatorial SAXS reflections with the spacing 36 Å are detected clearly even after the drawing 18


Page 18 of 43

Page 19 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

of the coiled coils. As suggested by Fraser and Parry32, the 36 Å spacing may correspond to the intermolecular distance between the neighboring four-stranded coils. The observation of this reflection in the cross-β-structure suggests the reservation of the bundles of four-stranded columns side-by-side. From these experimental data (i) and (ii), we strongly speculate that the transformation occurs by keeping the aggregation state of coiled four strands. That is to say, the morphology of the cross-β-sheets transformed from the four strands of coiled α-helices is four strands of coiled cross-β-sheets (The concrete picture of our suggestion is given in Figure 12). As another possibility, the angle 19o might come from the deformation of the originally-rectangular cell in the formation process of the cross-β-sheet from the α-helix. In this case, the shearing deformation might occur between the neighbouring columns. The real reason has not yet been known. Anyhow, we should remember that a series of the meridional SAXS peaks with the spacing 364 Å observed for the coiled α-helices disappear when the sample is drawn to 118% strain. These peaks correspond to the period of the coiled coil conformation. The disappearance of the reflections caused by the drawing suggests the deformation of the periodical coiled structure. The cross-β-structure is formed inside the individual coiled column (a picture of the single column is schematically depicted for the yellow-colored-strand in the coiled coil model shown in Figure 1) and the folded short segments are connected by the intramolecular hydrogen bonds. Application of an external tension to the sample may generate the shear stress, and the shearing of the coiled coils may cause the transformation from the α-helical to the β-zigzag conformations inside the each coiled column. At the same time, disordering may occur in the relative position of these columns with the four-stranded bundle shape. The columns consisting of the cross-β inner structure are considered to be strongly bundled due to the hydrophobic interactions, which might come from the 19


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

characteristic sequence of the amio acid units. A more detailed discussion of the role of the amino acid sequences will be described in the next section.

3.4.2. Stretching-induced crystallization of parallel-β -sheets Figure 10e shows the β200 peak turning up on the equator beginning at a stretching rate of 47% strain with continuously increasing intensity. The β200 peak is ascribed to the formation of a parallel-β-sheet structure, its appearance is consistent with the observation of the polarized 1625 cm-1 band in the ATR-FTIR spectrum (see Figure 9c). Three possible routes can be considered for the formation of the parallel-β-structure: (1) coiled α-helices → cross-β-sheet → parallel-β-sheet (2) straight α-helices → parallel-β-sheet (3) amorphous chains → parallel-β-sheet Case (1) - The parallel-β-sheet is assumed to be produced from the cross-β-sheet. However, it is difficult to imagine, however, that the molecular chain axis of the once-formed cross-βsheets turns into the meridional direction of the parallel-β-structure at a relatively small strain of 40%. Case (2) - The transition from the non-coiled straight α-helices to the parallel-β-sheet is considered a universal mechanism of protein unfolding.40 We have experimentally demonstrated that this transition is, in fact, attainable in regenerated Bombyx mori silkworm silk.37 However, in the present study the WAXD signal that proves the existence of non-coiled straight α-helices with the helical pitch at 5.4 Å was not detected through the whole deformational range. Hence, this process may also not be a dominant mechanism. Case (3) - Our starting silk material contains amounts of amorphous protein chains in addition to the helical regions. This, and the difficulties with the above two cases, support the route of 20


Page 20 of 43

Page 21 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

amorphous chain orientation and generation of a prallel-β-sheet structure by stretching beyond a strain of 50% (Figures 9 and 10).

3.5.

Relation between amino acid sequences and protein conformation

Sezutsu et al.41 have elucidated the hornet silk of Vespa simillima xanthoptera to be composed of four dominant proteins. These proteins are commonly composed of a central alanine-rich region and serine -rich N- and C-terminal regions as schematically shown in Figure 11a. The alanine-rich sequence is widely known to form the α-helical conformation as shown in Figure 11b. In the central alanine-rich region, the largest part consists of the heptad amino acid sequence-motif (a-b-c-d-e-f-g)n in which the positions a and d tend to be occupied by hydrophobic residues and the other positions by hydrophilic ones. This heptad motif is prone to form coiled coil structures as schematically shown in Figure 11c.8 The periodicity of 343.3 Å observed in natural hornet silk fiber agrees well with the length range 320-360 Å calculated for the coiled coil domain by Fraser and Parry32 using the number of amino acid residues included in the central heptad alanine sequence region of Vespa simillima xanthoptera.41 The periodicity of 364 Å determined for the regenerated silk gel film might be due to the dissolution treatment. Accordingly, the coiled α-helices in the gel film are probably formed by different combinations of the four helical proteins in the native fibers. The cross-β-sheet structure is linked to the same central heptad part of amino acid sequences (Figure 11c). As depicted in Figure 11c, serine-rich ends originally occur in the amorphous state in the unstretched gel film, but crystallize into parallel-β-sheets under the action of a tensile force. Serine-residues are well known β-sheet stabilizers41 and the hydroxyl group contributes to the formation of intermolecular hydrogen bonds.41,42

21


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 12 shows the whole image of the tension-induced transformation in the hornet silk gel film. The most important implication of the deduced structural transitions is that the coiled α-coil transforms to the cross-β-sheet structure, not to the parallel-β-sheet structure and besides it occurs by keeping the superstructure of four-strands (or four molecular chains). The orientation of the coiled α-helices existing in the central parts of the four strand proteins are enhanced by stretching. Parallel to that, the amorphous regions tend to change the orientation toward the stretching direction. The higher stress beyond some critical value causes the transition from the α to cross-β-structure in the central parts by keeping the four strand superstructure unchanged. (In fact, the SAXS data suggests the colied morphology of 364 Å period is deformed more or less but keeping the packing structure of 36 Å spacing corresponding to the four strands columnar structure.) On the other hand, in the serine-rich end parts, the regularization from the oriented amorphous to the parallel-β-sheet occurs a little later after the α to cross-β transition. Here, it should be mentioned that in our coiled cross-β-sheets model, one β-sheet is composed of a single chain folding with a β-turn-like manner as sketched in the (ii-c) and (iii-c). Thus, we call the single cross-β-sheet a single cross-β-strand as in the case of each coiled α-helical chain (refer to Figures 1 and 12). On the contrary, in the general models of amyloind fibril structure, as depicted by Sunde et al.43 or Nelson and Eisenberg44, each arrow sketched in the bottom (ii-c) and (iii-c) is often called a component β-strand, and therefore the single cross-β-sheet is regarded to be composed of a series of component β-strands closely perpendicular to the fibril long axis. With these descriptions in mind it becomes immediately clear that there is no essential difference between our proposed model of coiled cross-β-sheets and the amyloid fibril structure.43,44

22


Page 22 of 43

Page 23 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

4. CONCLUSIONS The tension-induced structural transition and its mechanism have been described for the gelfilm of regenerated hornet silk which was produced from the larva of the yellow hornet “Vespa simillima xanthoptera”. We have successfully revealed on the basis of detailed synchrotron SAXS/WAXD analyses and polarized FTIR spectroscopy that the original αhelices, organized in a four-stranded coiled coil superstructure, transform into cross-β-sheets arranged in the same superstructure, where the periodical structure of the coiled coils itself disappears but the 36 Å packing structure of four strands remains alive. The conservation of the four-strand superstructure is the result of the strong steric constraints against detwining. We further showed that, in parallel with the transformation process from α-helices to cross-βsheets, a stretching-induced crystallization of parallel-β-sheets occurs in the amorphous regions formed by the N- and C-terminal segments of the chains. These β-sheets are considered to form more or less straight fibrils with high chain orientation along the fibrillar axis. Our observation of the formation of an amyloid-like phase, that is the structural transformation process from the coiled-α-helices to coiled-cross-β-sheets, occurring in hornet silk not only sheds new light on the fiber formation process in insects but also has implications for the development of sophisticated biomimetic materials. Future studies are desirable to clarify the effects of the coexistence of two β-folding isomers in the stretched gel film on its mechanical properties.

ASSOCIATED CONTENT Supporting Information

23


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The following files are available free of charge. The SAXS profile of the native hornet silk (Vespa mandarinia japonica) (PDF); The ATR-FTIR spectra of the pressed and unpressed hornet silk gel-films (PDF); The results of the time-resolved synchrotron measurement during the wet-stretching of hornet silk gel-film (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (T.K.) *Email: [email protected] (A.K.S.) Notes

The authors declare no competing financial interest

ACKNOWLEDGMENTS The synchrotron radiation experiments were performed at the BL40B2 of SPring-8 with the approval of Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2014B1027, 2014B1487 and 2016A1440). T.K. is grateful for the financial support by JSPS KAKENHI (Grant Number 15K07803) and by JST-JICA, SATREPS program. K.T. is grateful for the financial support by MEXT “Strategic Project to Support the Formation of Research Bases at Private Universities (2010-2014 and 2015-2019)”. A.K.S. is grateful to the Japan Society for the Promotion of Science (JSPS) for their support by an Invitation 24


Page 24 of 43

Page 25 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

fellowship and a Bridge fellowship in 2013 and 2016, respectively. He wishes to thank Prof. H. KURATA, head of the Laboratory of Electron Microscopy and Crystal Chemistry, Kyoto University, for his kind hospitality, and his group members Dr. T. NEMOTO and Dr. T. OGAWA for their expert assistance in part of the experimental work. The authors also thank Mrs. Anne Galley, London, UK, for proof reading the manuscript.

REFERENCES (1) Asakura, T.; Ashida, J.; Yamane, T.; Kameda, T.; Nakazawa, Y.; Ohgo, K.; Komatsu, K. A repeated ß-turn structure in poly(Ala-Gly) as a model for silk I of Bombyx mori silk fibroin studied with two-dimensional spin-diffusion NMR under off magic angle spinning and rotational echo double resonance. J. Mol. Biol. 2001, 306, 291-305. (2) Asakura, T.; Umemura, K.; Nakazawa, Y.; Hirose, H.; Higham, J.; Knight, D. Some observations on the structure and function of the spinning apparatus in the silkworm Bombyx mori. Biomacromolecules 2007, 8, 175-181. (3) Rising, A.; Johansson, J. Toward spinning artificial spider silk. Nature Chem. Biol. 2015, 11, 309-315. (4) Lefèvre, Th.; Boudreault, S.; Cloutier, C.; Pézolet, M. Diversity of molecular transformations involved in the formation of spider silks. J. Mol. Biol. 2011, 405, 238253. (5) Van Beek, J.D.; Hess, S.; Vollrath, F.; Meier, B.H. The molecular structure of spider dragline silk: folding and orientation of the protein backbone. Proc. Natl. Acad. Sci. USA 2002, 99, 10266-10271. (6) Kameda, T.; Walker, A. A.; Sutherland, T. D. In Biotechnology of silk, 1st ed.; Asakura, T. and Miller, T.; Springer 2014, Chap. 5, 87-106. 25


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(7) Crick, F. H. C. Is α-keratin a coiled coil? Nature 1952, 170, 882-883. (8) Pauling, L.; Corey, R. Compound helical configurations of polypeptide chains: Structure of proteins of the α-keratin type. Nature 1953, 171, 59-61. (9) Lupas, A. N.; Gruber, M. The structure of α-helical coiled coils. Adv. Protein Chem. 2005, 70, 37-78. (10) Kameda, T.; Kojima, K.; Sezutsu, H.; Zhang, Q.; Teramoto, H.; Tamada, Y. Hornet (Vespa) silk composed of coiled-coil proteins. Kobunshi Ronbunshu 2010, 67, 641-653. (11) Atkins, E. D. T. A four-strand coiled-coil model for some insect fibrous proteins. J. Mol. Biol. 1967, 24, 139-141. (12) Kameda, T.; Nemoto, T.; Ogawa, T.; Tosaka, M.; Kurata, H.; Schaper, A. K. Evidence of

α-helical coiled coils and β-sheets in hornet silk. J. Struct. Biol. 2014, 185, 303-308. (13) Kameda, T.; Kojima, K.; Miyazawa, M.; Fujiwara, S. Film formation and structure of hornet silk. Z. Naturforsch., C: J. Biosci. 2005, 60, 906-914. (14) Kameda, T.; Kojima, K.; Togawa, E.; Sezutsu, H.; Zhang, Q.; Teramoto, H.; Tamada, Y. Drawing-induced changes in morphology and mechanical properties of hornet silk gel films. Biomacromolecules 2010, 11, 1009-1018. (15) Yin, J.; Chen, E.; Porter, D.; Shao, Z. Enhancing the toughness of regenerated silk fibroin film through uniaxial extension. Biomacromolecules 2010, 11, 2890-2895. (16) Parker, K. D.; Rudall, K. M. The silk of the egg-stalk of the green lace-wing fly. Nature 1957 179, 905-906. (17) Geddes, A. J.; Parker, K. D.; Atkins, E. D. T.; Beighton, E. “Cross-β” conformation in Proteins. J. Mol. Biol. 1968, 32, 343-358.

26


Page 26 of 43

Page 27 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(18) Kenchington, W. The larval silk of Hyperia SPP. (Colleoptera: Curculionidae). A new example of the cross-β protein conformation in an insect silk. J. Insect Physiol. 1983, 29, 355-361. (19) Walker, A. A.; Weisman, S.; Trueman , H. E.; Merritt , D. J.; Sutherland, T. D. The other prey-capture silk: Fibres made by glow-worms (Diptera: Keroplatidae) comprise cross-βsheet crystallites in an abundant amorphous fraction. Comp. Biochem. Physiol., B 2015, 187, 78-84. (20) Astbury, W. T.; Beighton, E.; Weibull, C. The structure of bacterial flagella. In: Fibrous Proteins and their Biological Significance. Symp. Soc. Exper. Biol. IX, Cambridge Univ. Press 1955, 282-305. (21) Kenney, J. M.; Knight, D.; Wise, M. J.; Vollrath, F. Amyloidogenic nature of spider silk. Eur. J. Biochem. 2002, 269, 4159-4163. (22) Andersson, M.; Chen, G.; Otikovs, M.; Landreh, M.; Nordling, K.; Kronquist, N.; Westermark, P.; Jörnvall, H.; Knight, S.; Ridderstråle, Y.; Holm, L.; Meng, Q.; Jaudzems, K.; Chesler, M.; Johansson, J.; Rising, A. Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains. PLoS Biol. 2014, 12, e1001921. (23) Poole, J.,; Church, J.S.; Woodhead, A.L.; Huson, M.G.; Sriskantha, A.; Kyratzis, I.L.; Sutherland, T.D. Continuous production of flexible fibers fom transgenically produced honeybee silk proteins. Macromol. Biosci. 2013, 13, 1321-1326. (24) Morris, K. L.; Rodger, A.; Hicks, M. R.; Debulpaep, M.; Schymkowitz, J.; Rousseau, F.; Serpell, L. C. Exploring the sequence-structure relationship for amyloid peptides. Biochem. J. 2013, 450, 275-283. (25) Chiti, F.; Dobson, C. M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 2006, 75, 333-366. 27


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(26) Kelly, J. W. Alternative conformations of amyloidogenic proteins govern their behavior. Curr. Opin. Struct. Biol. 1996, 6, 11-17. (27) Maury, C. P. J. The emerging concept of functional amyloid. J. Intern. Med. 2009, 265, 329-334. (28) Litvinov, R. I.; Faizullin, D. A.; Zuev, Y. F.; Weisel, J. W. The α-helix to β-sheet transition in stretched and compressed hydrated fibrin clots. Biophys. J. 2012, 103, 10201027. (29) Jahn, T. R.; Makin, O. S.; Morris, K. L.; Marshall, K. E.; Tian, P.; Sikorski, P.; Serpell, L. C. The common architecture of cross-β amyloid. J. Mol. Biol. 2010, 395, 717-727. (30) Parry, D. A. D.; Fraser, R. D. B.; Squire, J. M. Fifty years of coiled-coils and α-helical bundles: A close relationship between sequence and structure. J. Struct. Biol. 2008, 163, 258-269. (31) Busson, B.; Briki, F.; Doucet, J. Side-chains configurations in coiled coils revealed by the 5.15 Å meridional reflection on hard α-keratin X-ray diffraction patterns. J. Struct. Biol. 1999, 125, 1-10. (32) Fraser, R. D. B.; Parry, D.A.D. The molecular structure of the silk fibers from Hymenoptera aculeate (bees, wasps, ants). J. Struct. Biol. 2015, 192, 528-538. (33) Takahashi, T.; Gehoh, M.; Yuzuriha, K. Structure refinement and diffuse streak scattering of silk (Bombyx mori). Int. J. Biol. Macromol. 1999, 24, 127-138. (34) Sun, H. COMPASS: An ab initio force-field optimized for condensed-phase applicationsoverview with details on alkane and benzene compounds. J. Phys. Chem. B 1998, 102, 7338-7364.

28


Page 28 of 43

Page 29 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(35) Zandomeneghi, G.; Krebs, M. R. H.; Mccammon, M. G.; Fändrich, M. FTIR reveals structural differences between native β-sheet proteins and amyloid fibrils. Protein Sci. 2004, 13, 3314-3321. (36) Ridgley, D. M.; Claunch, E. C.; Barone, J. R. Characterization of large amyloid fibers and tapes with Fourier transform infrared (FT-IR) and Raman spectroscopy. Appl. Spectrosc. 2013, 67, 1417-1426. (37) Yoshioka, T.; Tashiro, K.; Ohta, N. Molecular orientation enhancement of silk by the hotstretching-induced transition from α-helix-HFIP complex to β-sheet. Biomacromolecules 2016, 17, 1437-1448. (38) Tashiro, K.; Yamamoto, H.; Yoshioka, T.; Ninh, T. H.; Tasaki, M.; Shimada, S.; Nakatani, T.; Iwamoto, H; Ohta, N.; Masunaga, H. Hierarchical structural change in the stress-induced phase transition of poly(tetramethylene terephthalate) as studied by the simultaneous

measurement

of

FTIR

spectra

and

2D

synchrotron

undulator

WAXD/SAXS/ data. Macromolecules 2014, 47, 2052-2061. (39) Takahashi, Y.; Sumita, I.; Tadokoro, H. Structural studies of polyesters. IX. Planar zigzag modification of poly(ethylene oxide). J. Polym. Sci. Polym. Phys. Ed. 1973, 11, 21132122. (40) Kreplak, L.; Doucet, J.; Dumas, P.; Briki, F. New aspects of the α-helix to β-sheet transition in stretched hard α-keratin fibers. Biophys. J. 2004, 87, 640-647. (41) Sezutsu, H.; Kajiwara, H.; Kojima, K.; Mita, K.; Tamura, T.; Tamada, Y.; Kameda, T. Identification of four major hornet silk genes with a complex of alanine-rich and serinerich sequences in Vespa simillima xanthoptera Cameron. Biosci. Biotechnol. Biochem. 2007, 11, 2725-2734.

29


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(42) Kameda, T.; Ohkawa, Y.; Yoshizawa, K.; Naito, J.; Ulrich, A. S.; Asakura, T. Hydrogenbonding structure in serine side chains in Bombyx mori and Samia Cynthia ricini silk fibroin determined by solid-state 2H NMR. Macromolecules 1999, 32, 7166-7171. (43) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. F. Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J. Mol. Biol. 1997, 273, 729-739. (44)Nelson, R.; Eisenberg, D. Structural models of amyloid-like fibrils. Adv. Protein Chem. 2006, 73, 235-282.

Figure 1 The differences of molecular chain conformation between (a) α-helix, (b) cross-βsheet, and (c) parallel-β-sheet. The parallel-β-structure model (c) was constructed based on the unit cell parameters proposed by Takahashi et al.33 (a = 9.38 Å, b = 9.49 Å, and c (fiber axis) = 6.98 Å, α = β = γ = 90.0°). The cross-β-structure model (b) was created by minimizing the Takahashi model with the chain folding, where the energy minimiation was performed using a COMPASS force field34 (a = 13.2 Å, b = 9.49 Å, and c (fiber axis) = 9.56 Å, α = 103.0°, β = 89.4°, γ = 93.4°).

30


Page 30 of 43

Page 31 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 2 (a) Experimental setup at the beamline 40B2 at SPring-8 for the simultaneous measurement of the stress-strain behavior and the time-resolved synchrotron X-ray scattering during wet-stretching of hornet silk gel films. A humidity chamber was attached to the Linkam tensile stage. (b) Schematic drawing of the general measurement geometry.

31


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3 Two-dimensional SAXS pattern (a) and meridional profile (b) of a bundle of native hornet silk fibers. Enlargements of selected parts of the lower end profile are inset in (b), a close-up of the higher-q-range is shown in (c) (see yellow colored rectangles in Figure 3(b)).

32


Page 32 of 43

Page 33 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 4 Two-dimensional WAXD patterns obtained (a) of the native hornet silk fiber bundle, and of the regenerated gel film (d) in through-view and (f) in edge-view orientation. The meridional and equatorial q-profiles (b, c) of the pattern in (a), the meridional profile (e) of pattern (d), and (g, h) of the pattern in (f) are placed below each respective pattern.

33


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5 Two-dimensional WAXD (a) and SAXS (b) patterns obtained from the un-stretched silk gel films in the through-view (left column) and edge-view (right columns), and from ε = 120% hot-stretched gel film (edge-view only). The meridional (c) and equatorial (d) q-profiles were scanned from the edge-view SAXS patterns.

34


Page 34 of 43

Page 35 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(a)

36.2Å

(b)

36.2Å

S.D. edge-view X-ray unstretched gel-film

stretched gel-film

Figure 6 Proposed changes of the structure deduced from the SAXS data (Figure 5) before and after stretching of the silk gel film. In the as-prepared film (a), the coiled coils (each coiled coil is expressed with a solid double-headed arrow and the long axis of coiled coil corresponds to the direction of the arrow) are arranged in plane-orientation with a layer distance of 36.2 Å (the β-sheet structures are not shown). In each layer, the coiled coils are arranged isotropically and this situation is schematically drawn using radially distributed arrows. After stretching (b), the coiled coil conformation disappears but the layer distance of the original plane orientation is retained. According to the WAXD analysis in Figure 4, the dominant structure changed from the coiled coil to two different kinds of oriented β-sheet structures, cross-β and parallel-β, represented by broken double-headed arrows. Both the long axes of the crystalline cross-β and parallel-β entities correspond to the direction of the arrow.

35


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7 (a) Through-, edge, and end-view two-dimensional WAXD patterns obtained for the 120% hot-stretched silk gel film, (b) equatorial and meridional 2θ-profiles of the edgeview pattern. (c) The deviation angle of the off-meridional β200 reflection was evaluated by peak separation analysis for the azimuthal profiles. (d) The totally rotated two-dimensional WAXD pattern (left) was obtained by rotating the film around the stretching axis. The cross-β and parallel-β diffraction features are illustrated in (d) (right). In the figures, the cross-β and parallel-β are represented as ×β and //β , respectively.

36


Page 36 of 43

Page 37 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 8 (a) Two dimensionl WAXD fiber diagram calculated from a crystal structure of the typical parallel-β-form proposed to the silk II crystalline modification of Bombyx mori silkworm silk,33 (b) the one from a crystal structure of the cross-β-form derived by rotating the structure in (a) around the b-axis, (c) the observed totally rotated one for the 100% stretched sample (same pattern shown in Figure 7(d)), and (d) the compound diagram of the calculated parallel- and cross-β-forms shown in (a) and (b). The X-ray diffraction patterns of the two structures were calculated using the commercial software Cerius2 (version 4.10, Biovia Ltd.). In the compound pattern (d), the colored diffraction spots belong to the cross-βstructure, the monochrome spots to the parallel-β structure. The compound pattern in (d) is in close qualitative agreement with the observed pattern (d).

37


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 9

(a) Schematic drawing of the experimental geometry of polarized-ATR-FTIR

spectroscopy and of the relationship between the direction of amide-I and ν(C=O) bonds in the two types of β-sheet (cross-β and parallel-β ) and the polarized IR beam (electric vectors E// and E⊥). The polarized (E//)-spectra (green color) and (E⊥)-one (red color) measured for the 38


Page 38 of 43

Page 39 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

hot-stretched gel films of ε = 0, 30, 60, and 100% are shown in panel (b), together with the unpolarized spectra (blue color). The absorbance ratio of the cross-β

over parallel-β

structures of the perpendicularly polarized bands is plotted versus strain in diagram (c).

Figure 10 (a) Changes of the 2D-WAXD patterns and (b) schematic drawings of the relevant scattering features obtained by time-resolved synchrotron WAXD measurements during wetstretching of the silk gel film. Panel (c) shows the q-profiles focused on the helical pitch of the coiled α-helix. In Figure 10(d) the azimuthal profiles of the 200 reflections show the 39


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

orientation details of the cross-β (×β )- and parallel-β (//β)-sheet structure. The corresponding intensity changes evaluated from (c) and (d) are plotted in panel (e). The upper part of panel (e) displays the stress-strain curve measured simultaneously during the wet-stretching process.

(a)

Ser-rich

Ser-rich

Aｌ-rich heptad motif (coiled-coil part)

(b) random coil

α-helix

random coil

four-strand aggregation

(c) amorphous

coiled-coil

amorphous

Figure 11 (a) Schematic representation of the general order of amino acid sequences in the protein chain of hornet silk. The proteins are composed of a central alanine-rich region and of Ser-rich N- and C-terminal regions. (b) The alanine-rich region in each protein is in the αhelix conformation, the terminal regions form a random coil or β-sheet structure in the asprepared gel film. (c) Each set of four α-helices is organized into a four-stranded coiled coil in natural as well as regenerated silk.

40


Page 40 of 43

Page 41 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 12 Proposed mechanisms (i) → (iii) and hierarchical structural models (a) → (d) of the tension-induced structural changes in a hornet silk gel film. In the as-prepared film (i), the randomly oriented four-strand coiled coils (i-a) are oriented in the plane of the film. In the coiled coil, each strand has an α-helical conformation (i-b) and the long axis of it tilts against the one of the coiled coil as schematically depicted in (i-c and d). At strains ranging from 20 to 50 % the coiled coils become gradually oriented in the stretching direction (ii-a). The coiled α-helices start to transform into cross-β (×β)-sheets at an almost perfect orientation level leading to coiled cross-β-sheets (ii-b). In the coiled cross-β-sheet, each cross-β-strand is speculated to be a β-turn-like conformation and the long axis of it tilts against the one of the coiled cross-β-sheet as depicted in the bottom of (ii-c and d). In addition, portions of parallel-

β (//β)-sheets appear when the stretching level exceeds 50% (iii-a), they are speculated to be originated from the oriented amorphous parts at the ends of the coiled coil segments. The helix-to-cross-beta transformation and the stretching of the resulting cross-β-structure continues up to strains ε ~ 120% without leading to a final parallel-β-sheet conformation. The red coloured broken line shows the intramolecular hydrogen bond in both the coiled α-helices and cross-β-sheets and the intermolecular one in the parallel-β-sheets. 41


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

42


Page 42 of 43

Page 43 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Biomacromolecules


Transformation of Coiled Î±-Helices into Cross-Î²-Sheets

Recommend Documents