Article pubs.acs.org/Macromolecules
Structural Determination of the Tandem Repeat Motif in Samia cynthia ricini Liquid Silk by Solution NMR Yu Suzuki,† Shuto Kawanishi,‡ Toshimasa Yamazaki,§ Akihiro Aoki,‡ Hitoshi Saito,∥ and Tetsuo Asakura*,‡ †
Tenure-Track Program for Innovative Research, University of Fukui, 3-9-1 Bunkyo, Fukui, Fukui 910-8507, Japan Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo 184-8588, Japan § National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan ∥ Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan ‡
ABSTRACT: The wild silkworm Samia cynthia ricini produces silk fibroin containing polyalanine sequences. The structure of a typical tandem repeat sequence YGGDGG(A)12GGAG within S. c. ricini silk fibroin in liquid silk was determined by solution NMR. 13C, 15N, and 1H shifts were assigned from solution NMR spectra for the tandem repeat. TALOS-N was then used to predict the backbone dihedral angles from the chemical shifts. Dihedral angles revealed a well-structured α-helix for the polyalanine region and less stable capping motifs for the N- and Cterminal regions. Consistent with this, the amide proton temperature coefficients of the last glycine in the N-terminal region and the first two glycines in the C-terminal region show more positive values than that of a random coil structure. Thus, the α-helical structure of the polyalanine region of S. c. ricini liquid silk is stabilized by capping motifs.
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main Ala CO and Ala Cα peaks shift to higher field; meanwhile, the main Ala Cβ peak shifts to lower field due to an increase in the random coil fraction. These are the same shift tendency observed by urea-induced helix−coil transition of S. c. ricini liquid silk.15,16 The proportion of helix component obtained from the NMR spectrum is consistent with that obtained from circular dichroism quantitatively.15 Solid state NMR was used to determine the dihedral angles and distribution of conformations in silk fibroin of S. c. ricini. The most probable conformation of polyalanine regions in natural silk fibers is an antiparallel β-sheet, although film produced from liquid silk appears to be primarily α-helical.17−20 A more precise structure of S. c. ricini silk fibroin before spinning was studied by a combination of site-specific stable isotope labeled 34-residue model peptides and several solid state NMR techniques.11,21,22 The dihedral angles of the central Ala residues in the polyalanine region and Gly residues adjacent to the N- and C-terminal side of the polyalanine region were determined. This study showed that the α-helical structure of the polyalanine region of silk fibroin from S. c. ricini tends to be highly wound at both terminal ends. Here, we aimed to determine the structure of the S. c. ricini silk fibroin repeat motif in native liquid silk. The determination of the atomic level structure of silk fibroin in liquid silk is
INTRODUCTION Silk fibroin produced by the domestic mulberry silkworm Bombyx mori is a promising biomaterial.1−3 However, in nature there are a variety of wild silkworms, each producing silk fibroin with a unique primary and higher order structure. Recently, these wild silkworm silks have received considerable attention as potentially valuable biomedical materials.4−6 Diverse mechanical properties and morphologies among a variety of wild silks are advantages to yield novel biomaterials with appropriate properties for specific application. Also, the functional sequence is presented in a primary structure of some wild silks, for example, wild silks containing Arg-Gly-Asp sequence which is characterized by the cell adhesion activity are studied for scaffold materials of tissue engineering. Samia cynthia ricini (S. c. ricini) is one such wild silkworm and the primary structure of its silk fibroin consists of tandemly repeated sequences made up of a polyalanine region and a Glyrich region.7 The primary structure of silk fibroin from S. c. ricini resembles that of spider dragline silk. However, silk fibroin from S. c. ricini has a longer polyalanine region ((A)10−14) than that of the dragline silk ((A)5−6).8−10 The most frequent sequence is YGGDGG(A)12GGAG. Before spinning to silk fiber, silk fibroin is stored in the silk gland as an aqueous solution, which is called “liquid silk”. The conformation of S. c. ricini liquid silk has been studied by 13C in vivo and solution NMR,11−16 and the polyalanine region gives a single peak due to rapid conformational exchange between the α-helix and random coil. As the temperature is increased, the © 2015 American Chemical Society
Received: August 2, 2015 Revised: August 31, 2015 Published: September 3, 2015 6574
DOI: 10.1021/acs.macromol.5b01717 Macromolecules 2015, 48, 6574−6579
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Macromolecules
Figure 1. (a) Assignments for the Ala, Gly, Tyr, and Asp peaks in the TOCSY spectrum and (b) sequential assignments in the HN/Hα and HN/HN regions of NOESY spectrum for the N-terminal part, Y1GGDGGA7 (left) and C-terminal part, A18GGAG22 of S. c. ricini liquid silk (right).
extremely challenging because S. c. ricini silk fibroin has an enormous molecular mass (227 kDa), and liquid silk is very viscous due to its high protein concentration (∼20% w/w). Recently, we clarified the structure of B. mori silk fibroin repeat motifs in liquid silk by applying a combination of multidimensional solution NMR with prediction of the backbone dihedral angles using the program TALOS-N.23 Here, we report a similar study on S. c. ricini fibroin. We achieved a sequential assignment of the most common S. c. ricini silk fibroin repeat motif, Y1GGDGG6(A)12G19GAG22, by 2D 1H NMR with the aid of 1H−15N and 1H−13C HSQC spectra. Using the chemical shifts for 1H, 13C, and 15N, we were able to derive the dihedral angles of each amino acid by employing the program TALOSN.24 From this information, we were able to build a model of the protein. Next, amide proton temperature coefficients were analyzed in order to verify the structure. Examination of the amide temperature coefficients strongly suggests that the polyalanine region forms an α-helix. Moreover, the N- and Cterminal regions form capping motifs, which at the C-terminus adopt a Schellman C-cap structure. The cap motifs are important because they contribute to the stability of the polyalanine α-helix. This is the first study to investigate the molecular structure of the repeat motif containing the Gly-rich region as well as the polyalanine portion of the protein for liquid silk.
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(Nippon Nosan Kogyo Co., Tokyo, Japan). The silk glands containing liquid silk were extracted from the S. c. ricini larvae after the fifth day of the fifth instar and soaked in a dish filled with distilled H2O so that the liquid silk could be gently removed from the silk glands.15 The liquid silk was placed in a 5 mm diameter NMR tube, and after 30 min the water was removed to eliminate sericin from the liquid silk surface. The concentration of the liquid silk is 18% (w/v) as reported previously.15 Next, the liquid silk in the NMR tube was placed together with a sealed capillary containing D2O for NMR locking. The liquid silk prepared for NMR measurements was stored at 4 °C until use and the NMR experiments were carried out within one or 2 days of sample preparation. NMR Measurements. All NMR experiments were carried out using a Bruker Avance750 spectrometer equipped with a 5 mm inverse triple-resonance probe head with three-axis gradient coils, operating at a 1H frequency of 750.13 MHz, 13C frequency of 188.64 MHz, and 15 N frequency of 76.02 MHz. A sealed capillary containing D2O was used for NMR locking. Chemical shifts were directly referenced to the H2O peak and indirectly referenced to TSP (trimethylsilylpropanoic acid) used as an external reference for 1H and 13C and to liquid NH3 for 15N. One-dimensional 1H NMR spectra were measured with WATERGATE for water peak suppression at intervals of 5 °C over a temperature range of 5−40 °C. Resonance assignments for the 1H signals were initially accomplished by 2D TOCSY and 2D NOESY experiments and confirmed by heteronuclear NMR experiments. 1H, 13 C, and 15N sequential resonance assignments were obtained by 2D 1 H−15N HSQC and 2D 1H−13C HSQC. NOESY and HSQC spectra were recorded at 10 °C. TOCSY spectra were recorded at intervals of 10 °C over a temperature range of 10−40 °C. NMR measurement parameters were as follows. NOESY was recorded with a mixing time
MATERIALS AND METHODS
Sample Preparation. Fertilized eggs of S. c. ricini were reared in our laboratory by feeding them an artificial diet, Silk Mate L4M 6575
DOI: 10.1021/acs.macromol.5b01717 Macromolecules 2015, 48, 6574−6579
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Macromolecules of 150 ms, 13.3 ppm spectral width in both t1 and t2 dimensions, 512 and 2048 complex points in t1 and t2 dimensions, 8 scans. TOCSY with a MLEV-17 mixing sequence was recorded with a mixing time of 70 ms, 13.3 ppm spectral width in both t1 and t2 dimensions, 512 and 2048 complex points in t1 and t2, four scans. 1H−13C HSQC was recorded with 165.7 ppm spectral width in t1 and 13.3 ppm in t2 dimensions, 128 and 2048 complex points in t1 and t2, 16 scans. 1 H−15N HSQC was recorded with 26.3 ppm spectral width in t1 and 13.3 ppm in t2 dimensions, 128 and 2048 complex points in t1 and t2, 64 scans. The NMR data were processed using NMRPipe,25 and the spectra were analyzed using SPARKY.26 Dihedral angle constraints for the main chain were derived from database analysis of the chemical shifts (13Cα, 13Cβ, 1Hα, 1HN, and 15N) of the backbone atoms using TALOS-N.24 Computational Conformation Analysis. To obtain a structural model, energy minimization was carried out using a semiempirical molecular orbital method. The MOPAC2012 software used for these calculations was based on Hamiltonian AM1. A structural model of the peptide YGGDGG(A)12GGAG was built with dihedral angles obtained from TALOS-N.
chemical shift values are summarized in Table 1. Although S. c. ricini silk fibroin contains many other flanking sequences at Table 1. 1H, 13C, and 15N Chemical Shifts for YGGDGG(A)12GGAG Obtained from Solution NMR Measurements of S. c. ricini Liquid Silk at 10 °C for HN, Hα, Cα, Cβ, and Na residue no. 1 2 3 4 5 6 7 8−17 18 19 20 21 22
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RESULTS Resonance Assignments for the Repeated Motif. The S. c. ricini silk fibroin consists of 93 tandem repeats of a polyalanine region flanked by glycine rich (Gly-rich) regions.7 The length of the polyalanine region varies from 10 to 14 residues, although the most common such region is made up of 12 residues. The total length of the Gly-rich region varies from 10 to 24 residues, which comprises mainly Gly along with a smaller proportion of Ala, Tyr, Asp, and Ser as well as other amino acids. A typical amino acid sequence from the Gly-rich region is YGGDGG, which appears 36 times at the N-terminal side of the polyalanine region. Likewise, the common sequence GGAG appears 65 times at the C-terminal side of the polyalanine region. Thus, the sequence Y1GGDGG6(A)12G19GAG22 can be used as a representative repeat sequence of S. c. ricini silk fibroin. Resonance assignments of 1H, 15N, and 13C resonances for the residues in the repetitive motif were obtained by comprehensive NMR analysis, using NOESY, TOCSY, 1 H−15N, and 1H−13C HSQC. Resonance assignments were first achieved for the NH chemical shifts of Ala, Gly, Tyr, and Asp by analyzing the TOCSY spectrum, as shown in Figure 1a. The peaks at around 8.0 and 8.3 ppm were both assigned to Ala with the former comprising a α-helix in the polyalanine region, while the latter adopts a random coil in the Gly-rich region. Next, sequential assignments for the N- and C-terminal parts of the repetitive motif sequence, which are adjacent to the polyalanine region, were accomplished by analyzing the HN/ Hα and HN/HN regions in the NOESY spectrum with the aid of TOCSY, 1H−15N HSQC, and 1H−13C HSQC. The assignment scheme for the N-terminal part, Y1G2G3D4G5G6A7, is described below as shown for the HN/Hα and HN/HN region spectra in Figure 1b, left. The sequential assignment was initiated from the NOE peak at 8.24/4.49 ppm for the NH/Hα resonance of Y1. The inter-residue cross peaks in the HN/HN region also aided the above assignments. In this manner, the 1H Hα/HN assignments for the amino acid residues in the YGGDGGA sequence were completed. Moreover, the 15N and 13 C chemical shifts were also assigned by 1H−15N and 1H−13C HSQC, and the consistency of the 1H chemical shift assignments was confirmed. Likewise, for the C-terminal region, A18G19G20A21G22, sequential assignments were performed as shown in Figure 1b, right. The 1H, 13C, and 15N
Tyr Gly Gly Asp Gly Gly Ala Ala Ala Gly Gly Ala Gly
HN
Hα
Cαb
Cβ
N
8.24 8.51 7.89 8.35 8.68 8.40 8.14 7.99 7.82 8.08 8.14 8.30 8.51
4.49 3.91 3.93 4.62 3.91 4.05 4.14 4.18 4.18 3.93 3.91 4.28 3.91
57.8 45.0 45.0 53.5 45.0 45.0 54.0 55.0 55.0 45.0 45.0 52.3 45.0
38.2
120.0 111.0 108.1 120.0 110.2 108.5 122.0 121.5 121.0 106.5 108.1 123.6 108.0
40.6
17.6 17.6 17.6
18.4
a Chemical shifts were referenced to TSP (ppm). bGly Cα shifts were overlapped and all have shifts in the range 45.0 ± 0.3.
lower abundance, the high frequency of the flanking YGGDGG and GGAG sequences at N- and C-terminal ends of the polyalanine sequence, respectively, means that it has been possible to assign this consensus sequence completely. Dihedral Angles Derived from the Chemical Shifts. The program TALOS-N is a database system for empirical prediction of backbone dihedral angles (ϕ, φ) with the highest possible accuracy using a combination of six types of backbone chemical shifts (1Hα, 13Cα, 13Cβ, 13CO, 15N, and 1HN) and sequence information.24 The TALOS-N program was applied to the YGGDGG(A)12GGAG motif using the 13Cα, 13Cβ, 1Hα, 1 HN, and 15N chemical shifts listed in Table 1 to obtain structural information. The predicted dihedral angles for each residue are plotted in Figure 2. The ϕ and φ angles of the polyalanine region are −62 ± 2° and −30 ± 11°, respectively. These values are strikingly similar to those of a typical α-helix (ϕ, φ) = (−60°, −45°). This result confirmed that the polyalanine region of S. c. ricini silk fibroin forms an α-helix in liquid silk. The dihedral angles of Nand C-terminal residues do not indicate typical secondary structure formation. To evaluate whether these residues are
Figure 2. Plot of dihedral angles for each residue of the S. c. ricini repeated motif in liquid silk derived from TALOS-N. ◆ and □ denote ϕ and ψ, respectively. Error bars show the estimated standard deviation provided by TALOS-N. 6576
DOI: 10.1021/acs.macromol.5b01717 Macromolecules 2015, 48, 6574−6579
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Macromolecules
hydrogen bonds are formed between CO of Gly6 and NH of Ala9 and between the CO of Gly6 and NH of Ala10. In addition, the dihedral angles of Gly6 (ϕ, φ) = (−79°, −179°) are close to the average dihedral angle values of the α-helix Ncap residue (−92 ± 24°, 167 ± 8°).29 These findings demonstrate that Gly6 serves as the N-cap like motif for the polyalanine α-helix.29 This is further supported by the presence of a type I β-turn structure from Gly3 to Gly6, which is consistent with the previous report that the N-cap residue frequently corresponds to the last residue of the turn.30 At the C-terminal region of the model structure, hydrogen bonds are formed between the CO of Ala16 and NH of Ala21 and between the CO of Ala17 and NH of Gly20, as shown in Figure 5. These hydrogen bond combinations are characteristic
well-defined or random coil, Wishart’s random coil index (RCI) was used.27 The RCI chart is shown in Figure 3. The RCI
Figure 3. RCI chart of the S. c. ricini repeated motif in liquid silk derived from Wishart’s random coil index.
values for the polyalanine residues are small, which means the polyalanine residues are well-defined. The structural disorder gradually increases with distance from polyalanine. This indicates that the residues neighboring the polyalanine segment have partially defined structures but the terminal residues are random coil. Next, a model structure was built and energy minimized based on the obtained dihedral angles using MOPAC shown in Figure 4. The polyalanine region forms an α-helical structure.
Figure 5. The C-terminal part of the structure forms intramolecular hydrogen bonds between Ala16 CO and Ala21 HN, Ala17 CO and Gly20 HN which are characteristic of the Schellman C-cap motif.
of the Schellman C-cap motif.29 Specifically, the Schellman Ccap motif is defined as a six-residue fragment that is located at the end of an α-helix and exhibits a double hydrogen bond pattern, namely NH of (i + 5) and CO of (i), NH of (i + 4) and CO of (i + 1). The six-residue fragment from Ala16 to Ala21 in the S. c. ricini model structure contains the same combination of hydrogen bonds as the Schellman C-cap motif. A further feature of the Schellman motif is the high probability of Gly at i + 4 because the backbone ϕ angle of i + 4 is positive. The typical ϕ angle of i + 4 in this motif is 73 ± 19°. The ϕ angle of Gly20, which is the i + 4 residue in the C-terminal part GGAG, is 99 ± 13°. This observation strongly indicates that the C-terminal part of the molecule forms a Schellman C-cap motif, which stabilizes the α-helical structure of the polyalanine region. Both the N- and C-terminal caps have a higher random coil population than the polyalanine region (Figure 3) but are well populated. Temperature Dependence of the Amide Proton Chemical Shift. A change in the amide proton chemical shift as a function of temperature can be used to give some indication of hydrogen bond strength or formation.31 TOCSY spectra of S. c. ricini liquid silk were measured at different temperatures from 10 to 40 °C in increments of 10 °C. The overlaid spectra of Hα/HN and Hβ/HN corresponding to the Ala region are shown in Figure 6. Amide proton chemical shifts of Ala in the Gly-rich region were shifted to higher field as the temperature increased, i.e., from 8.32 ppm at 10 °C to 8.14 ppm at 40 °C. In an aqueous solution of peptide or protein, the chemical shifts of most amide protons move to a higher field as the temperature is increased. This is normally explained as a weakening of the downfield shift that is the normal
Figure 4. An energy-minimized model structure of YGGDGG(A)12GGAG built from estimates of the backbone dihedral angles using the program TALOS-N.
In an α-helix, the first four NH groups and last four CO groups necessarily lack intrahelical hydrogen bonds and instead are often capped by alternative hydrogen bond partners.28 From a global survey among proteins of known structure, several capping motifs can be identified for the N- and Cterminal ends. At the N-terminal region of the model structure, 6577
DOI: 10.1021/acs.macromol.5b01717 Macromolecules 2015, 48, 6574−6579
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Macromolecules
temperature coefficient more positive than −4.5 ppb/K, it is very likely to be hydrogen bonded, while if it exchanges rapidly and has a temperature coefficient more negative than −4.5 ppb/K, it is not hydrogen bonded.32 At the C-terminal part, G19G20A21G22, coefficient values at Gly19 and Gly20 are more positive than anticipated for a random coil, although the values at Ala21 and Gly22 correspond to random coil. In the model structure, NH of Gly19 and Gly20 hydrogen bonds with CO of Ala15 and Ala17 in the polyAla region, respectively. Amide proton coefficient values at Gly19 and Gly20 are consistent with hydrogen bonds in the model structure. At the N-terminal end, the coefficient of Gly6 is more positive than that of random coil. NH of Gly6 forms a hydrogen bond with CO of Gly3 associated by a type I β-turn from Gly3 to Gly6. On the other hand, the coefficients of Tyr1 and Gly2 are close to random coil values. Gly3 has a much more positive value, −2.2 ppb/K. This observation can be explained by the close contact between the aromatic ring of Tyr and the amide proton of Gly. Merutka et al. reported that amide proton temperature coefficients for Gly5 in random peptides containing aromatic residues G1G2Ar3G4G5 (Ar: aromatic residue) were lowered to −3.2 ppb/K (Phe), −3.3 ppb/K (Tyr), and −0.1 ppb/K (Trp).31 Based on these results, it was proposed that there is either some degree of conformational preference for the orientation of the aromatic side chain toward the carbonyl terminus or an interaction with Gly5. We observed a higher field chemical shift of Gly3 (7.89 ppm) compared to Gly2 (8.51 ppm) in Y1G2G3D4G5G6, which could be explained by the ring current effect of the Tyr aromatic ring. Taken together, our analysis of the amide proton temperature coefficients support the model structure of the S. c. ricini repeated motif and suggest that the tyrosine residue commonly found in the N-terminal glycine-rich sequence may have a structural role in interacting with Gly3. Studies of Bombyx mori silk fibroin in liquid silk showed that the presence of Tyr residues destabilized the local β-turn structure and led to bends and turns. Under the fiber formation, Tyr residues stabilize the ends of the forming βsheet and strengthen the ends of the crystallites.33 Tyr residues in S. c. ricini silk fibroin were expected to have these functions as well.
Figure 6. HN/Hα and Ala HN/Hβ regions of TOCSY spectra for S. c. ricini liquid silk at 10, 20, 30, and 40 °C shown in red, blue, green, and yellow, respectively.
consequence of hydrogen bonding, which results from an increase in the average distance between hydrogen bond donor and acceptor.32 Next, the backbone amide proton temperature coefficients for individual residues in the YGGDGG(A)12GGAG motif were estimated by plotting the linear relationship of the amide proton chemical shift as a function of temperature from 5 to 40 °C at intervals of 5 °C. The temperature coefficients thus obtained are plotted in Figure 7. The coefficients of Ala and Gly
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DISCUSSION In nature, silk proteins are secreted and stored in glands prior to processing into fibers. Silkworms produce silk fibroin fiber from aqueous silk fibroin solution in a remarkable process by applying shear stress at the spinneret and applying tension, which is brought about by repeated drawing back of the silkworm’s head.34 Furthermore, the entire process of fiber formation occurs in an aqueous milieu at ambient temperature. As such, silk fiber processing is attractive as a model for developing a sustainable fiber processing technology. However, to fully understand the fibroin processing mechanism, it is necessary to determine the fibroin structure before and after spinning. In this study, we evaluated the structure of S. c. ricini silk fibroin before spinning in native liquid silk, which was extracted from the silk glands of the silkworm. The assignment of the 13 C, 15N, and 1H solution NMR spectra for the repetitive sequence motif, YGGDGG(A)12GGAG, was achieved, and the corresponding chemical shifts were obtained. Next, we used the program TALOS-N to predict the backbone dihedral angles from the chemical shifts for this motif. The obtained dihedral angles indicate (i) an α-helical structure for the polyalanine
Figure 7. Plot of amide proton temperature coefficients for the S. c. ricini repeat motif in liquid silk with those of B. mori as random coil references.
in B. mori liquid silk, which is known to be random coil at room temperature, are shown as references. In general, the coefficients of the backbone amide protons in random coil are rather large and negative (values
