Article Cite This: Macromolecules XXXX, XXX, XXX−XXX
Determination of Local Structure of 13C Selectively Labeled 47-mer Peptides as a Model for Gly-Rich Region of Nephila clavipes Dragline Silk Using a Combination of 13C Solid-State NMR and MD Simulation Tetsuo Asakura,* Akio Nishimura, and Yugo Tasei Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan S Supporting Information *
ABSTRACT: For the first time, we elucidate the complex structure of the Gly-rich regions in Nephila clavipes dragline silk through synergistic experimental and theoretical studies. First, the 13C selectively labeled 47-mer peptides selected from the glycine (Gly)rich region of N. clavipes dragline silk were synthesized. The 13C CP/ MAS NMR spectra were analyzed to determine the fractions of the conformations of individual Gly and Ala residues through 13C conformation-dependent chemical shifts and peak deconvolution. By comparing the 13C solid-state NMR spectra of several simple model peptides, the presence of 31 helix in the 47-mer peptides was disproved, and the (Ala)6 regions were shown to form β-sheet structure in the staggered arrangement. Although the fraction of β-sheet components tended to increase and the fraction of random coil component decrease toward both chain ends, significant change in the fractions was observed depending on the amino acid position. These results were successfully rationalized through molecular dynamics simulation.
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states by the combined X-ray diffraction and 13C CP/MAS NMR methods coupled with selective 13C isotope labeling of the Ala Cβ carbons.23,27,41 From deconvolution analyses of the line shapes of the Ala Cβ carbons in the 13C CP/MAS NMR spectra, the fractions of the rectangular and staggered packing arrangements have been determined as 49% and 51%, respectively, in the dry state and 40% and 60%, respectively, in the hydrated state for NCF and 62% and 38% in the dry state and 81% and 19% in the hydrated state for RSP. Thus, the packing structure changes significantly between the two spider silks and also between the two physical states. In contrast, the conformation of the Gly-rich region has been considered to be random coil. However, because there are significant amounts of the Gly-Ala-Gly sequence in this region, and poly(Ala-Gly-Gly) takes on the 31 helix form as reported by X-ray diffraction42 and 13C solid-state NMR methods,10 it is important to examine the presence of 31 helix in the Gly-rich region. Actually, the presence of 31 helix in the Gly-rich region of MaSp1 in spider dragline silk fibers has been frequently proposed from 13C solid-state NMR experiments.8,13,18,43−45 However, the details of the structure of the Gly-rich region are still lacking. One of the main reasons which make it difficult to clarify the local structure of the Gly-rich region is the presence of irregular amino acid sequences other than the Gly-Ala-Gly sequence. These irregular sequences result in large variations in the local conformation and molecular structure. Thus, it is difficult to clarify the local conformations of individual residues
INTRODUCTION Spider silks have attracted researchers in many fields because they are the toughest protein fibers known in nature and have mechanical properties that are attractive for applications where strength and toughness are critical.1−3 The dragline silk of the golden-orb weaver, Nephila clavipes (N. clavipes), has become the benchmark for the studies of silk fibers.4 It contains two structural proteins, designated spidroin 1 (MaSp1) and spidroin 2 (MaSp2).5 The dominant MaSpl protein can be described as a block copolymer consisting of polyalanine (poly-A) and Glyrich regions. The former region has been associated with high fiber strength, and the latter is a source of the high elasticity observed for the spider silk fiber. So far, the structure of spider silks has been studied with many spectroscopic techniques including nuclear magnetic resonance (NMR),6−27 Raman spectroscopy,28−32 and X-ray diffraction (XRD) analyses.33−40 These techniques have provided many insights into the molecular structure and dynamics of the spider silk proteins. However, a complete picture of the structure and dynamics of spider dragline silk at the molecular level is still lacking because of the complex and amorphous nature of the proteins. Thus far, the most detailed molecular level picture of the structure and dynamics of the silk has been obtained from NMR spectroscopy. For example, it has been clearly shown that poly-A region in the dragline silk fiber adopts antiparallel β-sheet structure (AP-β) on the basis of 13C conformation-dependent chemical shifts of Ala residues.6−11 Moreover, we have determined for the first time the detailed packing arrangements of the AP-β poly-A region in Nephila clavata fiber (NCF) and recombinant AP-β silk fiber with the sequences from Araneus diadematus (RSP) in dry and hydrated © XXXX American Chemical Society
Received: March 14, 2018 Revised: April 24, 2018
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DOI: 10.1021/acs.macromol.8b00536 Macromolecules XXXX, XXX, XXX−XXX
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Table 1. 47-mer Model Peptides with Typical Sequence of Nephila clavipes Dragline Silk Used for Solid-State NMR Analysisa (E)4(A)6GGAGQ15GGYGG20LGSQG25AGRGG30LGGQG35AG(A)6(E)4 (E)4(A)6G [2-13C]G12 [3-13C]A13 [1-13C]G14QGGYGGLGSQGAGRGGLGGQGAG(A)6(E)4 (E)4(A)6GGAGQGGYGGLGSQ [2-13C]G25 [3-13C]A26 [1-13C]G27RGGLGGQGAG(A)6(E)4 (E)4(A)6GGAGQGGYGGLGSQGAGRGGLGGQ [2-13C]G35 [3-13C]A36 [1-13C]G37(A)6(E)4 (E)4(A)6G [1-13C]G12A [15N]G14Q GGYGGLGSQG AGRGG LGGQGAG(A)6(E)4 (E)4(A)6GGA [1-13C]G14Q [15N]G16GYGGLGSQGAGRGGLGGQGAG(A)6(E)4 (E)4(A)6GGAGQG [1-13C]G17Y [15N]G19GLGSQGAGRGGLGGQGAG(A)6(E)4 (E)4(A)6GGAGQGGYG [1-13C]G20L [15N]G22SQGAGRGGLGGQGAG(A)6(E)4 (E)4(A)6GGAGQGGYGGLGSQGAGRG [1-13C]G30L [15N]G32GQGAG(A)6(E)4 (E)4(A)6GGAGQGGYGGLGSQGAGRGGLG [1-13C]G33Q [15N]G35AG(A)6(E)4 (E)4(A)6GGAGQGGYGGLGSQGAGRGGLGGQ [1-13C]G35A [15N]G37(A)6(E)4
b b b b b c c c c c b
a
The isotope-labeled positions were marked by bold. The peptides were dissolved in water because of the presence of (E)4 blocks. bSynthesized in this work. cSynthesized previously.20
directly from the spider silk fiber. We believe a better approach is to use sequential model peptides with selective stable-isotope labeling and to study them with solid-state NMR. In our previous papers,10,20,24,46−51 several 13C- and/or 15N-labeled model peptides with long sequences that faithfully reproduce specific sequences in the Gly-rich region of spider silks have been used to determine the local structure by the combined evaluation of the conformation-dependent 13C CP/MAS NMR chemical shifts, 2D spin diffusion 13C solid-state NMR, and rotational echo double resonance (REDOR) experiments. We have also combined solid-state NMR with molecular dynamics (MD) simulation in order to understand the local structures and structural changes of B. mori silk fibroins.52,53 The MD simulations can play a critical role in connecting experimental restraints with potentially plausible molecular structures, and therefore it has been successfully used to understand the structures and structural changes of silk fibroins in spider silks, including silk fiber formation mechanism.54−61 Thus, the combination of solid-state NMR and MD is a very powerful and useful analytical tool for the investigation of the local conformation of the Gly-rich region. In this paper, we synthesized 13C-labeled 47-mer peptides with the sequence (E)4(A)6GGAGQGGYGGLGSQGAGRGGLGGQGAG(A)6(E)4 as the sequential model peptide for the Gly-rich region of N. clavipes dragline silk together with the nonlabeled 47-mer peptide. These are the same sequence of the model peptides published previously20 and have two (E)4 blocks at both ends in order to make them water-soluble. The precipitation of the peptides in aqueous solutions was achieved by lowering the pH to 4.20,50,51 These peptides were designed to be a model for the Gly-rich sequence in N. clavipes dragline silk fiber after spinning. For the determination of the fractions of the conformations adopted by individual residues in the peptides, we prepared two groups of the peptides with different 13C-labeling patterns. One group consisted of three kinds of the peptides with three successive 13C-labeled amino acid residues, [2-13C]Gly12[3-13C]Ala13[1-13C]Gly14, [2-13C]Gly 25 [3- 13 C]Ala 26 [1- 13 C]Gly 27 or [2- 13 C]Gly 35 [3- 13 C]Ala36[1-13C]Gly37. Here the sequence Gly-Ala-Gly is a repeated unit of poly(Ala-Gly-Gly). Another group comprised seven kinds of [1-13C] and [15N]Gly-double-labeled 47-mer peptides with different labeled positions. Among them, five kinds of peptides had already been used for REDOR experiments to determine the interatomic distances of [1-13C] Gly and [15N]Gly residues in the peptides by changing the labeled positions.20 In this paper, we synthesized another two kinds of 47-mer peptides where both [1-13C]Gly12 and [15N]Gly14
residues or both [1-13C]Gly35 and [15N]Gly37 residues were labeled. The labeled positions were closer to both poly-A sequences than the previous 47-mer peptides used for the REDOR experiment. Thus, seven kinds of [1-13C] and [15N]Gly-double-labeled 47-mer peptides were employed to determine the local conformation of different Gly residues in the peptides. The conformation-dependent chemical shifts of 13 C and 15N nuclei were also utilized in the analysis.10,24,52−55 Furthermore, MD calculations were performed systematically to determine the local conformation of the individual residues in the Gly-rich region, (A)6GGAGQGGYGGLGSQGAGRGGLGGQGAG(A)6. Here, two (E)4 blocks at both termini were eliminated. The calculated fractions of several conformations were compared with those of the observed fractions. Thus, for the first time, we could study the local conformations of the Gly-rich regions sandwiched by poly-A sequences using both observed and calculated results. The synergistic experimental and theoretical studies of the conformation of the sequential model peptides made possible a detailed understanding of the complex structure of the Gly-rich regions in N. clavipes dragline silk fiber.
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MATERIALS AND METHODS
Syntheses of the Peptides. Three kinds of 13C-labeled 47-mer peptides with the sequence, (E)4(A)6GGAGQGGYGGLGSQGAGRGGLGGQGAG(A)6(E)4, were synthesized together with the nonlabeled peptide. The 13C-labeled positions were [2-13C]Gly12[3-13C]Ala13[1-13C]Gly14, [2-13C]Gly25[3-13C]Ala26[1-13C]Gly27, or [2-13C]Gly35[3-13C]Ala36[1-13C]Gly37. In addition, two kinds of [1-13C]Gly and [15N] Gly-double-labeled peptides, i.e., [1-13C]Gly12 and [15N]Gly14-double-labeled and [1-13C]Gly35 and [15N]Gly37double-labeled 47-mer peptides, were also synthesized. To give solubility to the peptides in water, four successive E units were attached at both the N- and C-termini. These peptides were synthesized by the F-moc solid phase method as described previously (Pioneer Peptide Synthesizer, PE. Biosystems, Co. Ltd.).20 Details are described in the Supporting Information, “Procedure” section. The purified by high-performance liquid chromatography and the purities of these peptides were checked by 13C solution NMR and IR to be more than 95%. The [2-13C]Gly, [3-13C]Ala, [1-13C]Gly, and [15N]Gly (each 99% enrichment) were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). The five kinds of 47-mer sequential peptides with different [1-13C] and [15N] Gly-doublelabeled positions synthesized previously20 were also used. All these peptides were summarized in Table 1. The aqueous solutions of these peptides were precipitated at pH = 4 and were collected by centrifugation (16 000 rpm, 10 min, at 4 °C) and dried. The simple peptides of AP-β (Ala)6, AP-β (Ala)7, (Ala-Gly-Gly)10 with 31 helix, (Ala-Gly)15 with Silk I and Silk II forms, and (AG)5A[15N]G12(AG)6 with Silk I and Silk II forms were synthesized previously.10,23,24 Thus, B
DOI: 10.1021/acs.macromol.8b00536 Macromolecules XXXX, XXX, XXX−XXX
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Figure 1. Arrangement of the 47-mer peptides before MD simulation. The four extended peptide chains were oriented in antiparallel forms within the planes and parallel between the planes. Three parallel planes were assumed. The intermolecular hydrogen bonding systems of poly-A blocks were set in a staggered packing arrangement. transferable intermolecular potential functions (TIP3P)65 with a concentration of 0.5 g/mL (5792 molecules) were randomly added to the system by using the packing optimization software, Packmol,66 keeping at least an intermolecular distance of 2 Å. Bond length and bond angle constraints of the SHAKE algorithm67 were applied for all bonds with hydrogen atoms to stabilize the system. During the initial run described in this paragraph, a spring constraint to the poly-A blocks was applied to tether it to its initial coordinate. First, an energy minimization with 1 fs time step was performed. Then, an isoenthalpic−isobaric (NPH) ensemble with a Langevin thermostat,68 1 fs time step, and a temperature of 300 K was performed for 2 ns to obtain a canonical velocity distribution of water molecules. Subsequently, an isothermal−isobaric (NPT) ensemble with 2 fs time step as a temperature equilibration was performed for 20 ps. After reaching the temperature equilibration, an annealing run was performed. The system was heated from 300 to 375 K over a period of 1 ns and then cooled to 300 K over a period of 1 ns. After the annealing run, an evaporation run was performed by a NPT ensemble in which the water molecules were removed by 12 molecules per 1 ps within 1 ns. After removing the water molecules, the Replica Exchange Molecular Dynamics (REMD) method69 was used to identify molecular equilibrium structures. Sixteen replicas of the system were simulated at temperatures between 300 and 375 K with an interval of 5 K to ensure proper sampling of the large conformational space. In the REMD, a temperature swap between adjacent ensembles was attempted every 2 ps, and then the swap attempts would alternate between odd and even pairings. Each attempted swap of temperatures as either accepted or rejected was based on a Boltzmann-weighted Metropolis criterion. Each replica was simulated for a total of 1 ns, corresponding to a total simulation time of 16 ns. The trajectory of the system during the REMD simulation was recorded every 1 ps. Cluster analysis was performed using visual molecular dynamics (VMD),70 with the quality threshold (QT) algorithm71 to identify the largest clusters. Representative final structures were selected as the structures included in the largest cluster of the lowest temperature replica. The averaged fraction of several conformations of the individual residues in the Gly-rich region was calculated in VMD using the STRuctural IDEntification (STRIDE) algorithm,72 which used the Kabsch and Sander rules.73
in total, ten kinds of 47-mer peptides and seven kinds of simple peptides were used for solid-state NMR measurements. 13 C and 15N CP/MAS NMR Observation. Solid-state 13C and 15N CP/MAS NMR spectra of these peptides were recorded on Chemagnetics CMX-400 NMR and Bruker AVANCE 400 spectrometers at the 13C and 15N resonance frequencies of 100.6 and 40.5 MHz, respectively. Typical experimental parameters for the CP/MAS NMR experiments were 3.6 μs for 1H 90° excitation pulse, ramped CP pulse with a contact time of 2 ms, TPPM 1H decoupling pulse of 70 kHz during acquisition, and 9.0 kHz magic angle spinning rate. The 13C and 15N chemical shifts were indirectly calibrated using the signal of adamantane methylene to 28.8 ppm relative to TMS and ammonium chloride (15NH4Cl) to 18.0 ppm relative to ammonium nitrate as external reference, respectively. A total of 2048−20 480 scans were collected over a spectral width of 40 kHz with a recycle delay of 4 s. Lorentzian line broadenings of 40 or 20 Hz were applied before Fourier transformation for 47-mer peptides and others, respectively. The 13C CP/MAS NMR spectra of ten kinds of 47-mer peptides were deconvoluted by using Gaussian line shapes to determine the fraction of specific conformations. In the deconvolution of the spectra, the halfheight widths for the individual components of the peaks were changed while the chemical shifts were fixed.27,41 The error of the fraction values in the deconvolution analysis were about ±2% after several deconvolution trials. MD Simulation. MD simulations were performed for the sequence, (A)6GGAGQGGYGGLGSQGAGRGGLGGQGAG(A)6, selected from the Gly-rich region of N. clavipes dragline silk where the N and C termini were capped with acetyl group (Ace) and Nmethylamide group (NMe), respectively. The sequence was essentially the same as that used for the solid-state NMR observation except for the absence of two (E)4 blocks. The Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) molecular dynamics program62 and the Optimized Potentials for Liquid Simulations-all atom (OPLS-AA) force field63 were used for the MD simulation. Moreover, Lennard-Jones and long-range Coulombics pairwise interactions were used with a cutoff of 10 Å. Figure 1 shows the arrangement of the 47-mer peptides before MD simulation. The four extended peptide chains were oriented in antiparallel forms within the planes and parallel between the planes. Three parallel planes were assumed. The poly-A blocks were set in a staggered packing arrangement from the assignment of abovementioned 13C solid-state NMR spectrum. This structure was optimized with density functional theory (DFT) calculation.64 Additional intersheet distance of 6 Å was adopted to the poly-A blocks. To prevent image interactions, a periodic box wrapped each model by at least 5 Å. Water molecules compatible with the
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RESULTS C CP/MAS NMR Spectra of Nonlabeled 47-mer Peptide and Simple Peptides for Conformation-Dependent 13C Chemical Shift References. Figures 2a shows C
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DOI: 10.1021/acs.macromol.8b00536 Macromolecules XXXX, XXX, XXX−XXX
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of simple peptides (summarized in Table 2) were used for conformational analyses of individual Gly and Ala residues in the 47-mer peptides. Table 2. 13C CP/MAS NMR Chemical Shifts of Several Peptides and Bombyx mori Silk Fibroin Having Well-Defined Conformations 13
C chemical shift (ppm)
Ala sample (Ala)6 (antiparallel β-sheet structure in rectangular packing arrangement) (Ala)7 (antiparallel β-sheet structure: in staggered packing arrangement) (AG)15 (Silk I: type II βturn) (AG)15 (Silk II: mainly antiparallel β-sheet structure) (AGG)10 (31-helix) B. mori silk fibroin in water (random coil)
Cβ
Gly
Cα
CO
20.6
48.9
171.4
Cα
CO
19.8, 22.8
48.5
171.3
16.7
50.9
176.8
43.5
169.9
19.7, 22.6
48.7
171.9
42.4
168.0, 169.1
17.4 16.6
49.0 50.0
174.8 175.5
41.5 42.7
171.2 171.7
In Figure 2a, the chemical shift, 51.8 ppm, assigned to Glu Cα peak clearly suggested that two (Glu)4 sequences at both ends of the 47-mer peptides took on the AP-β structure.79 Therefore, the broad peak at about 30 ppm could be assigned to the Cβ and Cγ peaks of Glu residues with AP-β.79 The peaks at 20.3 ppm (main peak) and 22.7 ppm (shoulder peak) could be assigned to Ala Cβ carbons of two poly-A sequences located at both sides of Gly-rich region. It may be noted that the line shapes of the Ala Cβ carbons were similar to that of Ala Cβ carbons of (Ala)7 with AP-β in the staggered packing arrangement (Figure 2c) rather than that of (Ala)6 in the rectangular packing arrangement (Figure 2b), and therefore the two poly-A sequences were considered to take on the AP-β structure with staggered packing arrangement. The peak at 48.8 ppm in Figure 2a could be assigned to Ala Cα carbon which also supported the AP-β structure for poly-A sequences. The chemical shift of Gly Cα peak was observed at 42.7 ppm, which was close to the random coil chemical shift (Figure 2g) and different from that of the 31 helix (Figure 2f). In the carbonyl region of nonlabeled peptide, it was difficult to discuss the conformation of each residue because the carbonyl carbon peaks of all residues were observed. Thus, it was necessary to do selective stable-isotope labeling of the peptides in Gly-rich region. 13 C CP/MAS NMR Spectra of Three 13C-Labeled 47mer Peptides Incorporating Selective 13C-Labeled Block, [2-13C]Gly[3-13C]Ala[1-13C]Gly, in Different Positions. In order to study the local structures of Ala and Gly residues in the Gly-rich region, three kinds of 13C-labeled 47-mer peptides with three 13C-labeled contiguous amino acid residues in different positions were synthesized. Thus, three kinds of 13Clabeled blocks, [2-13C]Gly12[3-13C]Ala13[1-13C]Gly14, [2-13C]Gly 25 [3- 13 C]Ala 26 [1- 13 C]Gly 27 , or [2- 13 C]Gly 35 [3- 13 C]Ala36[1-13C]Gly37, were introduced into three 47-mer peptides. Here the sequence, Gly-Ala-Gly is a repeated unit of poly(AlaGly-Gly). Figures 3a−d show the expanded 13C CP/MAS NMR spectra of nonlabeled and three kinds of 13C-labeled peptides. Only 13C-labeled positions are marked in Figures 3b−
Figure 2. 13C CP/MAS NMR spectrum of (a) nonlabeled 47-mer peptides after precipitation of the aqueous solution at pH = 4. 13C CP/ MAS NMR spectra of simple peptides, (b) (Ala)6 with antiparallel βsheet (rectangular packing arrangement), (c) (Ala)7 with antiparallel β-sheet (staggered packing arrangement), (d) (Ala-Gly)15 with Silk I (type II β-turn) form, (e) (Ala-Gly)15 with Silk II form (antiparallel βsheet with small amounts of random coil), and (f) (Ala-Gly-Gly)10 with 31 helix. (g) The random coil chemical shifts of B. mori silk fibroin (mainly Gly and Ala residues) in aqueous solution are also shown as stick spectra.78 The characteristic chemical shifts of the model peptides are marked by arrows.
the 13C CP/MAS NMR spectrum of nonlabeled 47-mer peptide after precipitation of the aqueous solution at pH = 4. Only 13C solid-state NMR peaks from Ala, Gly, and Glu residues could be observed in the spectrum because of the large number of these residues. The 13C solid-state NMR spectra of simple peptides having well-defined conformations were also observed to show clearly the correlation between the 13C chemical shifts and conformations for Ala and Gly residues. Thus, Figures 2b−f are the 13C CP/MAS NMR spectra of (b) (Ala)6 with AP-β in rectangular packing arrangement, 23(c) (Ala)7 with AP-β in staggered packing arrangement,23,64 (d) (Ala-Gly)15 with Silk I (Type II β-turn) form,52,53,74,75 (e) (AlaGly)15 with Silk II form (AP-β with small amounts of random coil),53,64,76,77 and (f) (Ala-Gly-Gly)10 with 31 helix.10 In addition, 13C random coil chemical shifts of the Gly and Ala residues of B. mori silk fibroin in aqueous solution are shown as a stick spectrum (g) for comparison.78 The 13C chemical shifts D
DOI: 10.1021/acs.macromol.8b00536 Macromolecules XXXX, XXX, XXX−XXX
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region, the 13C chemical shift of single and sharp Gly37 CO peak was 168.0 ppm, which was in agreement with that of the higher field shoulder peak of the Gly CO carbon of (AlaGly)15 with AP-β (Figure 2e). Thus, we observed two peaks with different chemical shifts, 169.1 and 168.0 ppm, for the Gly CO carbon which was already observed previously.77 As for the assignments of these two peaks, we will revisit them in the Discussion section. The small peak at 171.7 ppm came from natural abundance CO peak (Figure 3a), and therefore the Gly37 residue took on exclusively the AP-β structure. This was due to the influence of poly-A sequence with AP-β. The Gly14 and Gly27 CO peaks were asymmetric, and the chemical shift position of the main peak was in good agreement with that of random coil. 13 C CP/MAS NMR Spectra of 13C and 15N DoubleLabeled 47-mer Peptides Systematically Incorporating the [1-13C]GlyX[15N]Gly Block in Different Positions. Figure 4 shows a comparison of 13C CP/MAS NMR spectra
Figure 3. 13C CP/MAS NMR spectra of 13C successively triple-labeled 47-mer peptides incorporated as the [2-13C]Gly[3-13C]Ala[1-13C]Gly block in different positions: (a) nonlabeled 47-mer peptides, (b) [2-13C]Gly12[3-13C]Ala13[1-13C]Gly14 block, (c) [2-13C]Gly25[3-13C]Ala26[1-13C]Gly27 block, and (d) [2-13C]Gly35[3-13C]Ala36[1-13C]Gly37 block. The asterisk shows mainly the Ala Cα, Cβ, and CO peaks of two (Ala)6 sequences.
d. On the basis of the 13C chemical shift (48.8 ppm) of Ala Cα peak marked by asterisks for natural abundance Ala residues (Figure 3a), the poly-A regions on these peptides took on the AP-β structure. These peptides seemed to be appropriate models to study the local conformations in N. clavipes dragline spider silk fiber. It was clear that the Ala13, Ala26, and Ala36 residues in the Gly-Ala-Gly sequences in Gly-rich region did not adopt the 31 helix because the Cβ chemical shifts of these three Ala residues are not 17.4 ppm which is a chemical shift reference of poly(Ala-Gly-Gly) with 31 helix (Figure 2f). The line shapes of [3-13C]Ala Cβ carbons were similar between two labeled carbons of Ala13 and Ala26 residues but quite different from the Ala36 peak (Figures 3b−d). Thus, the Ala36 residue adopted mainly the AP-β structure with only a small amount of random coil. On the other hand, the Ala13 and Ala26 β peaks consisted of both random and AP-β, and the fraction of the former was higher. This seemed reasonable for the Ala26 residue because the C-terminal poly-A sequence was expected to have less influence on the AP-β conformation because the Ala26 residue was located close to the center of the Gly-rich region, and as a result, the conformation was expected to be random coil. However, the influence of the N-terminal poly-Α sequence with AP-β decreased rapidly for the Ala13 residue although there were two Gly residues, Gly11 and Gly12, between Nterminal poly-A sequence and the Ala13 residue. Because the amount of 13C conformation-dependent chemical shifts of Gly Cα carbons was considerably smaller compared with those of Ala Cβ and Gly CO carbons,74 it seemed difficult to deduce the local conformation from the Gly Cα chemical shifts in detail. However, three Gly12, Gly25, and Gly35 Cα peaks were observed at a lower field than the Gly Cα peak of the 31 helix observed at 41.5 ppm (Figure 2f). These Gly residues did not adopt the 31 helix as mentioned above as the three Ala residues in the Gly-rich region. In the Gly CO
Figure 4. 13C CP/MAS NMR spectra of 13C and 15N double-labeled 47-mer peptides systematically incorporated as the [1-13C]GlyX[15N]Gly block in different positions. The 13C chemical shifts of main Ala Cβ and Ala Cα peaks (broken lines) were around 20 and 48.8 ppm, respectively. In the Gly 13CO region, the conformational analysis was performed by using the chemical shifts of β-sheet (broken line), βturn (thick dotted line), and random coil (thin dotted line) in Figure 2.
of seven kinds of 47-mer peptides incorporating the different [1-13C]GlyX[15N]Gly blocks as summarized in Table 1. Only the 13C-labeled Gly positions are marked in Figure 4. In all the spectra, the 13C chemical shifts of main Ala Cβ and Ala Cα peaks were around 20.3 and 48.8 ppm, respectively, confirming that the conformations of these seven peptides on poly-A sequences adopted the AP-β structure. Thus, these seven peptides serve as appropriate models of the local conformation of N. clavipes dragline spider silk fiber. In Figure 4, the Gly 13 CO peak was dominant among all carbonyl carbons, and therefore we can discuss the difference in the local conformation of the Gly residue depending on the position in the Gly-rich region. The line shape of Gly14 CO peak was essentially the same as that of Gly14 CO peak of the [2-13C]Gly12[3-13C]Ala13[1-13C]Gly14 block in the tripleE
DOI: 10.1021/acs.macromol.8b00536 Macromolecules XXXX, XXX, XXX−XXX
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was already confirmed from the chemical shift of [1-13C]Gly37 peak (Figure 3d). The same chemical shift of B. mori silk fibroin with Silk II form was reported previously.80 Thus, the chemical shift, 85.6 ppm, seemed to be a marker for the AP-β structure. However, 15N chemical shifts also depend on the preceding amino acid residues significantly. For example, as shown in Figure S1c, the random coil 15N chemical shift of Gly residue of B. mori silk fibroin in aqueous solution was 87.6 ppm for the AlaGly sequence but 90.3 ppm for the SerGly sequence.81 Thus, the chemical shift difference of 15N Gly residues between AlaGly and SerGly blocks was 2.7 ppm, which was relatively large compared with the magnitude of conformation-dependent chemical shifts. These data suggested that it was better to use conformation-dependent 13C chemical shift for the conformational analysis in this paper rather than the complicated 15N chemical shift. Indeed, the difference in random coil 13C chemical shift of Gly carbonyl peak between GlyAla and GlySer sequences in B. mori silk fibroin in aqueous solution was 0.4 ppm, sufficiently small compared with the conformationdependent 13C chemical shifts.82 Fraction of Different Conformations of Individual Gly and Ala Residues in Gly-Rich Regions of 13C-Labeled 47mer Peptides Determined by Peak Deconvolution. Peak deconvolution of 13C CP/MAS NMR spectra of ten kinds of 13 C-labeled peptides is shown in Figure 6. Figure 6a shows three [3-13C]Ala and three [1-13C]Gly carbons of three kinds of peptides incorporating three 13C-labeled blocks in different positions. Deconvolution of three Gly 13Cα peaks was not attempted because the amount of the 13C conformationdependent chemical shifts was considerably smaller. Moreover, seven kinds of [1-13C]Gly carbons in the peptides with different 13 C-labeling positions are shown in Figure 6b. Peak deconvolution of Ala Cβ peaks in Figure 6a was performed by assuming the presence of random coil, β-turn, and AP-β with different packing structures separated into two peaks: A (19.8 ppm) and B (22.8 ppm).24,25,41,76 For the peak deconvolutions of Gly CO peaks in Figures 6a and 6b, the presence of similar conformations was assumed except for one main peak at 169.1 ppm for AP-β. In both cases of the deconvolutions of Ala Cβ and Gly CO peaks, the presence of β-turn conformation was required to obtain better fits in some cases. The fraction values from deconvolution are summarized in Table 3. Figure 7 is the plot of the fractions of several conformations against the position of the 13C-labeled nuclei in a 47-mer peptide chain. The fraction determined from [1-13C]Gly14 peak was similar between two kinds of the peptides within experimental error. This signifies high reproducibility of the data shown here. In general, the residues in Gly-rich region seemed to take on basically the random coil conformation because of the presence of many Gly residues with a high degree of freedom of the backbone bonds. However, because of the presence of poly-A sequences with AP-β at both N- and C-termini, the residues located close to the poly-A sequences tended to adopt the AP-β structure. Actually, the fraction of AP-β rapidly decreased toward the center from the edge, in contrast to that of random coil as shown in Figure 7. Thus, the change in the fractions of individual residues in the Gly-rich region became roughly symmetric. The fraction of β-turn structure tended to decrease after increasing once toward the center from the edge although the change was relatively small. Of course, the details of the change would be modulated depending on the primary structure. Interesting results were shown as small increases in the fraction values of AP-β at the Ala26 and Gly27 residues. This
labeled 47-mer peptide shown in Figure 3b. This signifies high reproducibility of the experiment data. It may be noted that the line shapes of Gly12 and Gly35 CO carbons were clearly different from other Gly CO peaks. Because the 169.1 ppm peak could be assigned to AP-β (Figure 2e), there were significant amounts of AP-β contained in these two residues. This seemed reasonable because these two Gly residues were closer to poly-A sequences with AP-β than other Gly residues. Between these two peaks, the fraction of AP-β in the Gly35 residue was larger than that of the Gly12 residue. For a more detailed discussion about the fractions of all the conformations, deconvolution of these peaks is required and will be done later. 15 N CP/MAS NMR Spectra of 13C and 15N DoubleLabeled 47-mer Peptides Systematically Incorporating the [1-13C]GlyX[15N]Gly Block in Different Positions. Figure 5 shows a comparison of 15N CP/MAS NMR spectra
Figure 5. 15N CP/MAS NMR spectra of 13C and 15N double-labeled 47-mer peptides systematically incorporated as the [1-13C]GlyX[15N]Gly block in different positions. Only three chemical shift positions are marked, but conformational analysis using 15N chemical shift seems difficult as described in the text.
of seven kinds of 47-mer peptides with different [1-13C]GlyX[15N]Gly blocks. Only the 15N-labeled Gly positions are marked in Figure 5. Although the 15N conformation-dependent chemical shift has been reported, it was not easy to use 15N chemical shift for the conformational analysis in this paper.80 The reason is as follows. As shown in Figure S1 (Supporting Information), (a) the 15N CP/MAS NMR spectrum of (AG)5A[15N]G12(AG)6 with Silk I form showed a single sharp peak at 88.1 ppm, but (b) that of the Silk II form was asymmetric and the chemical shift of main peak at 88.1 ppm was in agreement with the Silk I peak (a). The 15N chemical shift of the shoulder peak at higher field (about 84 ppm) was close to that of the relatively sharp [15N]Gly37 peak observed at 85.6 ppm. The Gly37 residue took on the AP-β structure which F
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Table 3. Fractions of Several Conformations of 47-mer Peptides Determined from Deconvolution of the 13C CP/ MAS NMR Spectraa fraction (%) A13 A26 A36 G14 G27 G37 G12 G14 G17 G20 G30 G33 G35
β-sheet
random coil
β-turn
15 (9:6) 15 (11:4) 85 (53:32) 17 15 92 37 11 1 1 8 18 41
65 78 15 67 79 8 49 73 88 89 76 68 59
20 7 0 16 6 0 14 16 11 10 16 14 0
a The fractions of two different β-sheet structures of Ala residues are shown in parentheses as (β-sheet A:β-sheet B).
Figure 7. Plot of the fractions determined by the peak deconvolution of 13C CP/MAS NMR spectra of 47-mer peptide (Table 3) against the position of 13C-labeled nuclei in the Gly-rich region. Details are discussed in the text.
13
13
13
simulated peptides for further structural analysis, we tried to use the STRIDE algorithm. However, the STRIDE algorithm, which is based on the hydrogen-bonding criterion, tended to overestimate the fraction of AP-β when extended and bridge assigned to β-sheet because the residues of silk proteins readily formed hydrogen bonds even in random coil conformation. Actually, as shown in Figure S2a, the fraction of β-sheet was calculated to be very high for every residue in the Gly-rich region, and this did not match with the observed results. Therefore, we corrected the results of the STRIDE algorithm by using a criterion that limited the number of the residues in the β-sheet when we applied the calculated results to conformational analysis of silk chains. Specifically, we assigned a new residue to the β-sheet structure only if the residue was assigned as extended or bridge by the STRIDE algorithm and had a consecutive assignment of extended or bridge. By changing the number of consecutive residues, n, we plotted the fraction of β-sheet against the residue number in Figures S2b− e. As a result, when n was equal to 5, we could reproduce the observed results. The calculated fractions of several conformations of the representative structures of the REMD simulation are summarized in Table 4 and shown in Figure 8a for the case of n = 5. In order to compare the simulated results with the observed results (Figure 7), the calculated data which
14
Figure 6. Peak deconvolutions of (a) [3- C]Ala and [1- C]Gly carbons or [3-13C]Ala36 and [1-13C]Gly37 carbons of two kinds of 3C successively triple-labeled 47-mer peptides. (b) [1-13C]Gly carbons of seven kinds of [1-13C]Gly-47-mer peptides with different 13C-labeling positions. The deconvolutions were performed for the difference peaks by subtracting the peaks of natural abundance carbons from the 13Clabeled peaks. The observed (black lines) and calculated (purple broken lines) peaks are shown together with the deconvoluted peaks (red and yellow: β-sheet; green: β-turn; and blue: random coil).
was not the influence of C- and N-terminal of poly-A sequences with AP-β. By careful checking the primary structure of the Glyrich region, we noticed that the sequence from the 24 to 28 residue was Gln24Gly25Ala26Gly27Arg28, with no GlyGly sequences. Thus, the presence of two successive GlyGly residues as seen in the other region of the Gly-rich sequence of 47-mer peptide seemed to originate from the random coil conformation because of significant flexibilities of the backbone of the Gly residue. About 15% AP-β fraction was obtained from the deconvolution of [3-13C]Ala26 and [1-13C]Gly27 peaks. Calculation of the Fraction of Several Conformations of Individual Residues in the Gly-Rich Region from MD Simulation. In order to identify the conformations of the G
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corresponded to the observed residues were selected and are shown in Figure 8b. The typical image of the MD simulations is shown for the sequence (A)6GGAGQGGYGGLGSQGAGRGGLGGQGAG(A)6 in Figure S3. Almost all residues in the poly-A regions were in the AP-β structure. In the Gly-rich region, random coil conformation generally dominated; however, the fractions of several conformations varied with the residue number. There was a notable tendency that the fraction of AP-β had minimum values at positions of 17 and 32 and increased in value toward each terminus and also at the middle of the Gly-rich region, Gly25Ala26Gly27. On the other hand, the fraction of the random coil tended to increase from both terminus to the center of the Gly-rich region. The fraction of AP-β in the sequence in the C-terminal region, Gly35Ala36Gly37 was slightly larger than that in the sequence in the N-terminal region, Gly12Ala13Gly14. The fraction of βturn had an interesting tendency that it increased from both sides toward Gly14 and Gly32 and then decreased toward the center of the Gly-rich region. As for helix conformations, only 0.2% of the 31 helix conformation was found for the sequence of Gln12Gly13Gly14 sampled from the whole REMD trajectory of the lowest temperature replica, and no helix conformations were found in the representative structures.
Table 4. Fractions of Several Conformations Determined by the STRIDE Algorithm from the Results of MD Simulations fraction (%) G11 G12 A13 G14 Q15 G16 G17 Y18 G19 G20 L21 G22 S23 Q24 G25 A26 G27 R28 G29 G30 L31 G32 G33 Q34 G35 A36 G37
β-sheet
random coil
β-turn
84 60 16 16 14 7 1 1 5 12 11 11 13 12 13 20 21 17 14 11 2 1 15 23 24 63 75
14 24 55 50 65 74 76 72 90 77 82 83 80 83 80 73 72 73 76 78 77 70 71 69 76 37 25
2 16 29 34 21 19 23 27 5 11 7 6 7 5 7 7 7 10 10 11 21 29 14 8 0 0 0
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DISCUSSION The Gly-rich regions in spider dragline silk fiber are considered to be responsible for the high elasticity of the spider silk fiber.1,3,4 Therefore, it is important to elucidate the amino acid conformation in this region, but it has been difficult because of the presence of irregular amino acid sequences. We believe that a good approach is to study sequentially longer model peptides where the individual residues in the Gly-rich region are stableisotope labeled.10,20,24,46−51 The local conformation of each residue can then be discerned from the conformationdependent NMR chemical shifts of the isotope-labeled sites as shown in Figure 2.74,83−85 In the conformational analysis using conformation-dependent chemical shifts, we observed two peaks with different chemical shifts: 169.1 ppm (main peak) and 168.0 ppm (shoulder peak) in the Gly CO carbon of (Ala-Gly)15 with Silk II form (Figure 2e). Both peaks could be assigned to AP-β. For Ala Cβ of (Ala-Gly)15 with Silk II form, we could also observe two peaks: A (main peak) and B (shoulder peak) as assigned previously. These were both AP-β peaks, but the intermolecular arrangements were different as shown in Figure 9. On the basis of relative peak intensities of both Gly CO and Ala Cβ peaks (Figure 2e), the peak at 169.1 ppm (main peak) could be assigned to peak A and 168.0 ppm (shoulder peak) to peak B. Actually, the intermolecular arrangement around these two Gly carbonyl carbons was quite different and might be the origin of chemical shift difference for these two peaks as shown in Figure 9. Through peak deconvolution, we could determine the fractions of different conformations of individual residues in the Gly-rich region as summarized in Table 3 and Figure 7, showing a mixture of several conformations for each residue. Moreover, we disproved the presence of the 31 helix in the Gly-rich region from our results, as discussed later. MD simulation was used to examine the local conformations of the individual residues in the Gly-rich region and to compare the calculated and the observed results. In previous papers,45,55,56 it was impossible to check the calculated fractions of individual residues in detail because of the lack of observed
Figure 8. Plots of the fractions determined by the STRIDE algorithm, for the representative structure of the REMD simulation with the lowest temperature replica: (a) whole residue numbers and (b) residue numbers observed by 13C-labeled peptides.
H
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Therefore, it may be necessary to revisit the findings of previous papers. The presence of 31 helix in the Gly-rich region has been proposed from spectral simulation of 2D spindiffusion experiments of [1-13C]Gly-labeled Nephila madagascariensis dragline silk fiber.8 The spectral pattern on the basis of 13 C chemical shift tensor values of the Gly carbonyl carbons was calculated to reproduce the observed pattern by assuming a single conformation such as random coil, planar β-sheet, αhelical, and 31 helix conformation. Among them, 31 helix was the best fitted conformation to interpret the observed spectral pattern when one single conformation was assumed. However, the theoretical patterns calculated by assuming a mixture of βsheet and random coil conformations also seemed to reproduce the observed pattern. Actually, in their latter paper,13 the static [1-13C]Gly spectral pattern of the oriented samples from [1-13C]Gly-N. edulis dragline silk fiber showed a mixture of powder pattern (random coil) and the angle-dependent oriented pattern rather than one single conformation. The fraction of the latter oriented peak was 61% and that of random coil 39%. In the structural analysis, the [1-13C]Gly peak position changed significantly depending of the angle between the fiber direction of the sample and external magnetic field. The behavior of the angle-dependent peak position seemed similar between [1-13C]Gly and [1-13C]Ala peaks. In this work, the latter peak from poly-A sequences took on the AP-β structure, and therefore 61% of the Gly residue seemed also to take on AP-β. Bonev et al.17 also reported the static [1-13C]Gly spectral pattern of oriented [1-13C]Gly-N. edulis dragline silk fiber. The fraction of the oriented peak was 35%, and that of the powder pattern was 65%. Eles and Michal14 determined the fraction of the well-oriented [1-13C]Gly residues in the Gly-rich region of the oriented N. clavipes dragline silk fiber to be 47% and that of the other conformation to be polyglycine II although the latter calculation could not be taken as evidence for its existence. In addition, they also showed the fraction of the oriented components decreased in the relaxed state of the fibers. Thus, their structural analysis showed the presence of a mixture of powder pattern and angle-dependent oriented pattern rather than one single conformation. We reported the angle-dependent 13C and 15N solid-state NMR spectra of the oriented [15N]- or [1-13C]Ala- and [1-13C]Gly- B. mori silk fibroin fibers and determined the torsion angles of Ala and Gly residues in the backbone chain.86,87 The fraction of the oriented components was about 80%, and the other component which gave the powder pattern was about 20% for both residues. The torsion angles (ϕ, φ) determined for Ala and Gly residues in the oriented components were (−140°, 142°) and (−139°, 135°), respectively, indicating that these oriented residues took on the AP-β structure. Similar angle-dependent NMR analysis was applied for the oriented [15N]- or [1-13C]Ala- and [1-13C]GlyS. c. ricini silk fibroin fibers whose primary structure is similar to the spider dragline silk fiber, i.e., tandem repeats of poly-Ala and Gly-rich regions.88 The fraction of the oriented peak was 75%, and that of the powder pattern was 25% for Ala residues. On the other hand, the fraction of the oriented peak was 65%, and that of the powder pattern was 35% for Gly residue. Because the length of the poly-A region is 12 or 13, longer than those of spider dragline silk fiber (where the average length is 6 or 7), a lower fraction of AP-β of the Gly-rich region is deduced for the spider silk. Thus, the weight of the data seems to prefer a mixture of AP-β and random coil forms rather than one 31 helix form in the Gly-rich region in the spider dragline silk fiber.
Figure 9. Difference in the intermolecular distances (Å) from the red circled Gly CO carbons to two adjacent Gly NH hydrogens and two adjacent Gly CO oxygens, in configurations of (a) Silk II A and (b) Silk II B.89
data. Thus, only the averaged fractions of several conformations calculated by MD simulation could be compared with the observed data, and they interfered with the detailed conformational behavior of the Gly-rich region. Thus, for the first time we have been able to compare the local conformations of the Gly-rich regions between the observed and calculated results by MD simulation. In order to identify the local conformations, we tried to use the STRIDE algorithm. However, the STRIDE algorithm tended to overestimate the fraction of β-sheet present. Therefore, we corrected the result of the STRIDE algorithm by a criterion that limited the number of the residues in the β-sheet. The fraction of β-turn had an interesting tendency that increased at a short distance of 4−6 residues and decreased toward the center of Gly-rich region. This tendency was also observed in the experimental result. Figures 3 and 4 clearly shows that the local conformation of Gly residues close to both (Ala)6 sequences tended to form AP-β due to the strong influence of (Ala)6 with AP-β. The presence of 31 helix in the Gly-rich region of MaSp1 in spider dragline silk fibers has been proposed frequently8,13,18,19,43−45 because there are significant amounts of the sequences Gly-Ala-Gly, and poly(Ala-Gly-Gly) takes 31 helix as reported by X-ray diffraction42 and 13C solid-state NMR methods.10 This conclusion has been obtained mainly from the solid-state NMR analysis of 13C-labeled spider dragline silk fiber. However, our present 13C solid-state NMR data disprove the presence of the 31 helix in the Gly-rich region. I
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CONCLUSION In this work we have managed to determine the fractions of individual residues in the Gly-rich region using 13C-labeled 47mer peptides and 13C conformation-dependent chemical shifts together with peak deconvolution. The MD simulations have reproduced the observed results. Thus, for the first time, we can compare the local conformations of the Gly-rich regions sandwiched by poly-A sequences between the observed and calculated results. Synergistic experimental and theoretical investigations on the conformations of appropriate sequential model peptides have made possible a detailed understanding of the complex structure of the Gly-rich regions in N. clavipes dragline silk fiber. In addition, through these analyses, we have disproved the presence of the 31 helix conformation in the Glyrich region.
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REFERENCES
(1) Blackledge, T. A.; Pérez-Rigueiro, J.; Plaza, G. R.; Perea, B.; Navarro, A.; Guinea, G. V.; Elices, M. Sequential Origin in the High Performance Properties of Orb Spider Dragline Silk. Sci. Rep. 2012, 2, 782−787. (2) Asakura, T.; Miller, T. Biotechnology of Silk; Asakura, T., Miller, T., Eds.; Biologically-Inspired Systems; Springer Netherlands: Dordrecht, 2014; Vol. 5. (3) Lang, G.; Neugirg, B. R.; Kluge, D.; Fery, A.; Scheibel, T. Mechanical Testing of Engineered Spider Silk Filaments Provides Insights into Molecular Features on a Mesoscale. ACS Appl. Mater. Interfaces 2017, 9, 892−900. (4) Tokareva, O.; Jacobsen, M.; Buehler, M.; Wong, J.; Kaplan, D. L. Structure−function−property−design Interplay in Biopolymers: Spider Silk. Acta Biomater. 2014, 10, 1612−1626. (5) Sponner, A.; Schlott, B.; Vollrath, F.; Unger, E.; Grosse, F.; Weisshart, K. Characterization of the Protein Components of Nephila Clavipes Dragline Silk. Biochemistry 2005, 44, 4727−4736. (6) Simmons, A.; Ray, E.; Jelinski, L. W. Solid-State 13C NMR of Nephila Clavipes Dragline Silk Establishes Structure and Identity of Crystalline Regions. Macromolecules 1994, 27, 5235−5237. (7) Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Molecular Orientation and Two-Component Nature of the Crystalline Fraction of Spider Dragline Silk. Science 1996, 271, 84−87. (8) Kümmerlen, J.; van Beek, J. D.; Vollrath, F.; Meier, B. H. Local Structure in Spider Dragline Silk Investigated by Two-Dimensional Spin-Diffusion Nuclear Magnetic Resonance. Macromolecules 1996, 29, 2920−2928. (9) Michal, C. A.; Jelinski, L. W. Rotational-Echo Double-Resonance in Complex Biopolymers: A Study of Nephila Clavipes Dragline Silk. J. Biomol. NMR 1998, 12, 231−241. (10) Ashida, J.; Ohgo, K.; Komatsu, K.; Kubota, A.; Asakura, T. Determination of the Torsion Angles of Alanine and Glycine Residues of Model Compounds of Spider Silk (AGG)10 Using Solid-State NMR Methods. J. Biomol. NMR 2003, 25, 91−103. (11) Yang, Z.; Liivak, O.; Seidel, A.; LaVerde, G.; David, B.; Zax, A.; Jelinski, L. W. Supercontraction and Backbone Dynamics in Spider Silk: 13C and 2H NMR Studies. J. Am. Chem. Soc. 2000, 122, 9019− 9025. (12) Kishore, A. I.; Herberstein, M. E.; Craig, C. L.; Separovic, F. Solid-State NMR Relaxation Studies of Australian Spider Silks. Biopolymers 2002, 61, 287−297. (13) 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. U. S. A. 2002, 99, 10266−10271. (14) Eles, P. T.; Michal, C. A. A DECODER NMR Study of Backbone Orientation in Nephila Clavipes Dragline Silk under Varying Strain and Draw Rate. Biomacromolecules 2004, 5, 661−665. (15) Eles, P. T.; Michal, C. A. Strain Dependent Local Phase Transitions Observed during Controlled Supercontraction Reveal Mechanisms in Spider Silk. Macromolecules 2004, 37, 1342−1345. (16) Hronska, M.; van Beek, J. D.; Williamson, P. T. F.; Vollrath, F.; Meier, B. H. NMR Characterization of Native Liquid Spider Dragline Silk from Nephila Edulis. Biomacromolecules 2004, 5, 834−839. (17) Bonev, B.; Grieve, S.; Herberstein, M. E.; Kishore, A. I.; Watts, A.; Separovic, F. Orientational Order of Australian Spider Silks as Determined by Solid-State NMR. Biopolymers 2006, 82, 134−143. (18) Holland, G. P.; Jenkins, J. E.; Creager, M. S.; Lewis, R. V.; Yarger, J. L. Solid-State NMR Investigation of Major and Minor Ampullate Spider Silk in the Native and Hydrated States. Biomacromolecules 2008, 9, 651−657. (19) Holland, G. P.; Creager, M. S.; Jenkins, J. E.; Lewis, R. V.; Yarger, J. L. Determining Secondary Structure in Spider Dragline Silk by Carbon−Carbon Correlation Solid-State NMR Spectroscopy. J. Am. Chem. Soc. 2008, 130, 9871−9877. (20) Yamaguchi, E.; Yamauchi, K.; Gullion, T.; Asakura, T. Structural Analysis of the Gly-Rich Region in Spider Dragline Silk Using Stable-
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b00536. Procedure: details of peptide synthesis. (Figure S1) 15N CP/MAS NMR spectrum of (AG)5A[15N]G(AG)6 with (a) Silk I and (b) Silk II forms; (c) the random coil chemical shifts of B. mori silk fibroin in aqueous solution are shown as stick spectra;81 (Figure S2) different corrections of β-sheet fractions determined by the STRIDE algorithm, for the representative structure of the REMD simulation with the lowest temperature replica; a residue assigned to extended or bridge by the STRIDE algorithm is assigned to random coil conformation, if the residue has an equal or less number of consecutive residues than (a) 1 (no correction), (b) 2, (c) 3, (d) 4, and (e) 5; (Figure S3) typical images of MD simulations in this paper are shown for the sequence (A)6GGAGQGGYGGLGSQGAGRGGLGGQGAG(A)6 of N. clavipes dragline silk; (a) shows only backbone structure and (b) full structure including backbone and side chain; both sides in the sequence indicate antiparallel β-sheet structure of the sequences (Ala)6 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*(T.A.) Tel & Fax 81-42-383-7733; e-mail
[email protected]. jp. ORCID
Tetsuo Asakura: 0000-0003-4472-6105 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS T.A. acknowledges support by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Supports of Japan (JP26248050) and Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT). T.A. also acknowledges Dr. H. N. Cheng (Southern Regional Research Center, USDA, Agricultural Research Service, New Orleans, LA 70124) for discussions. J
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Article
Macromolecules Isotope Labeled Sequential Model Peptides and Solid-State NMR. Chem. Commun. 2009, 0, 4176−4178. (21) Jenkins, J. E.; Creager, M. S.; Butler, E. B.; Lewis, R. V.; Yarger, J. L.; Holland, G. P. Solid-State NMR Evidence for Elastin-like β-Turn Structure in Spider Dragline Silk. Chem. Commun. 2010, 46, 6714− 6716. (22) Jenkins, J. E.; Creager, M. S.; Lewis, R. V.; Holland, G. P.; Yarger, J. L. Quantitative Correlation between the Protein Primary Sequences and Secondary Structures in Spider Dragline Silks. Biomacromolecules 2010, 11, 192−200. (23) Asakura, T.; Okonogi, M.; Horiguchi, K.; Aoki, A.; Saito, H.; Knight, D. P.; Williamson, M. P. Two Different Packing Arrangements of Antiparallel Polyalanine. Angew. Chem., Int. Ed. 2012, 51, 1212− 1215. (24) Asakura, T.; Suzuki, Y.; Nakazawa, Y.; Yazawa, K.; Holland, G. P.; Yarger, J. L. Silk Structure Studied with Nuclear Magnetic Resonance. Prog. Nucl. Magn. Reson. Spectrosc. 2013, 69, 23−68. (25) Asakura, T.; Suzuki, Y.; Nakazawa, Y.; Holland, G. P.; Yarger, J. L. Elucidating Silk Structure Using Solid-State NMR. Soft Matter 2013, 9, 11440−11450. (26) McLachlan, G. D.; Slocik, J.; Mantz, R.; Kaplan, D.; Cahill, S.; Girvin, M.; Greenbaum, S. High-Resolution NMR Characterization of a Spider-Silk Mimetic Composed of 15 Tandem Repeats and a CRGD motif. Protein Sci. 2009, 18, 206−216. (27) Tasei, Y.; Nishimura, A.; Suzuki, Y.; Sato, T. K.; Sugahara, J.; Asakura, T. NMR Investigation about Heterogeneous Structure and Dynamics of Recombinant Spider Silk in the Dry and Hydrated States. Macromolecules 2017, 50, 8117−8128. (28) Shao, Z.; Vollrath, F.; Sirichaisit, J.; Young, R. J. Analysis of Spider Silk in Native and Supercontracted States Using Raman Spectroscopy. Polymer 1999, 40, 2493−2500. (29) Sirichaisit, J.; Brookes, V. L.; Young, R. J.; Vollrath, F. Analysis of Structure/Property Relationships in Silkworm (Bombyx Mori) and Spider Dragline (Nephila Edulis) Silks Using Raman Spectroscopy. Biomacromolecules 2003, 4, 387−394. (30) Rousseau, M.-E.; Lefèvre, T.; Beaulieu, L.; Asakura, T.; Pézolet, M. Study of Protein Conformation and Orientation in Silkworm and Spider Silk Fibers Using Raman Microspectroscopy. Biomacromolecules 2004, 5, 2247−2257. (31) Lefèvre, T.; Rousseau, M.-E.; Pézolet, M. Protein Secondary Structure and Orientation in Silk as Revealed by Raman Spectromicroscopy. Biophys. J. 2007, 92, 2885−2895. (32) Dionne, J.; Lefèvre, T.; Auger, M. Major Ampullate Spider Silk with Indistinguishable Spidroin Dope Conformations Leads to Different Fiber Molecular Structures. Int. J. Mol. Sci. 2016, 17, 1353. (33) Lucas, F.; Shaw, J. T. B.; Smith, S. G. Comparative Studies of Fibroins: I. The Amino Acid Composition of Various Fibroins and Its Significance in Relation to Their Crystal Structure and Taxonomy. J. Mol. Biol. 1960, 2, 339−349. (34) Work, R. W.; Young, C. T. The Amino Acid Compositions of Major and Minor Ampullate Silks of Certain Orb-Web-Building Spiders (Araneae, Araneidae). J. Arachnol. 1987, 15, 65−80. (35) Grubb, D. T.; Jelinski, L. W. Fiber Morphology of Spider Silk: The Effects of Tensile Deformation. Macromolecules 1997, 30, 2860− 2867. (36) Riekel, C.; Müller, M.; Vollrath, F. Situ X-Ray Diffraction during Forced Silking of Spider Silk. Macromolecules 1999, 32, 4464−4466. (37) Riekel, C.; Madsen, B.; Knight, D.; Vollrath, F. X-Ray Diffraction on Spider Silk during Controlled Extrusion under a Synchrotron Radiation X-Ray Beam. Biomacromolecules 2000, 1, 622− 626. (38) Glišović, A.; Salditt, T. Temperature Dependent Structure of Spider Silk by X-Ray Diffraction. Appl. Phys. A: Mater. Sci. Process. 2007, 87, 63−69. (39) Madurga, R.; Blackledge, T. A.; Perea, B.; Plaza, G. R.; Riekel, C.; Burghammer, M.; Elices, M.; Guinea, G.; Pérez-Rigueiro, J. Persistence and Variation in Microstructural Design during the Evolution of Spider Silk. Sci. Rep. 2015, 5, 14820.
(40) Munro, T.; Putzeys, T.; Copeland, C. G.; Xing, C.; Lewis, R. V.; Ban, H.; Glorieux, C.; Wubbenhorst, M. Investigation of Synthetic Spider Silk Crystallinity and Alignment via Electrothermal, Pyroelectric, Literature XRD, and Tensile Techniques. Macromol. Mater. Eng. 2017, 302, 1600480. (41) Asakura, T.; Tasei, Y.; Aoki, A.; Nishimura, A. Mixture of Rectangular and Staggered Packing Arrangements of Polyalanine Region in Spider Dragline Silk in Dry and Hydrated States As Revealed by 13C NMR and X-ray Diffraction. Macromolecules 2018, 51, 1058−1068. (42) Lotz, B.; Keith, H. D. The Crystal Structures of poly(Ala-GlyGly-Gly)II and poly(lAla-Gly-Gly)II. J. Mol. Biol. 1971, 61, 195−200. (43) Jenkins, J. E.; Sampath, S.; Butler, E.; Kim, J.; Henning, R. W.; Holland, G. P.; Yarger, J. L. Characterizing the Secondary Protein Structure of Black Widow Dragline Silk Using Solid-State NMR and X-Ray Diffraction. Biomacromolecules 2013, 14, 3472−3483. (44) Shi, X.; Holland, G. P.; Yarger, J. L. Molecular Dynamics of Spider Dragline Silk Fiber Investigated by 2H MAS NMR. Biomacromolecules 2015, 16, 852−859. (45) Gray, G.; van der Vaart, A.; Guo, C.; Jones, J.; Onofrei, D.; Cherry, B.; Lewis, R.; Yarger, J.; Holland, G. Secondary Structure Adopted by the Gly-Gly-X Repetitive Regions of Dragline Spider Silk. Int. J. Mol. Sci. 2016, 17, 2023−2035. (46) Asakura, T.; Yang, M.; Kawase, T. Structure of Characteristic Sequences in Nephila Clavipes Dragline Silk (MaSp1) Studied with 13C Solid State NMR. Polym. J. 2004, 36, 999−1003. (47) Asakura, T.; Yang, M.; Kawase, T.; Nakazawa, Y. 13C Solid-State NMR Study of Structural Heterogeneity in Peptides Containing Both Polyalanine and Repeated GGA Sequences as a Local Structural Model of Nephila Clavipes Dragline Silk (Spidroin 1). Macromolecules 2005, 38, 3356−3363. (48) Yang, M.; Nakazawa, Y.; Yamauchi, K.; Knight, D. P.; Asakura, T. Structure of Model Peptides Based on Nephila Clavipes Dragline Silk Spidroin (MaSp1) Studied by 13C Cross Polarization/magic Angle Spinning NMR. Biomacromolecules 2005, 6, 3220−3226. (49) Ohgo, K.; Kawase, T.; Ashida, J.; Asakura, T. Solid-State NMR Analysis of a Peptide (Gly-Pro-Gly-Gly-Ala)6-Gly Derived from a Flagelliform Silk Sequence of. Biomacromolecules 2006, 7, 1210−1214. (50) Yazawa, K.; Yamaguchi, E.; Knight, D. P.; Asakura, T. 13C SolidState NMR Study of the 13C-Labeled Peptide, (E)8GGLGGQGAG(A)6GGAGQGGYGG as a Model for the Local Structure of Nephila Clavipes Dragline Silk (MaSp1) before and after Spinning. Biopolymers 2012, 97, 347−354. (51) Yazawa, K.; Yamaguchi, E.; Aoki, A.; Nakazawa, Y.; Suzuki, Y.; Asakura, T. A Two-Dimensional Spin-Diffusion NMR Study on the Local Structure of a Water-Soluble Model Peptide for Nephila Clavipes Dragline Silk (MaSp1) before and after Spinning. Polym. J. 2012, 44, 913−917. (52) Yamane, T.; Umemura, K.; Asakura, T. The Structural Characteristics of Bombyx Mori Silk Fibroin before Spinning as Studied with Molecular Dynamics Simulation. Macromolecules 2002, 35, 8831−8838. (53) Yamane, T.; Umemura, K.; Nakazawa, Y.; Asakura, T. Molecular Dynamics Simulation of Conformational Change of poly(Ala-Gly) from Silk I to Silk II in Relation to Fiber Formation Mechanism of Bombyx Mori Silk Fibroin. Macromolecules 2003, 36, 6766−6772. (54) Ma, B.; Nussinov, R. Molecular Dynamics Simulations of Alanine Rich Beta-Sheet Oligomers: Insight into Amyloid Formation. Protein Sci. 2002, 11, 2335−2350. (55) Keten, S.; Buehler, M. J. Nanostructure and Molecular Mechanics of Spider Dragline Silk Protein Assemblies. J. R. Soc., Interface 2010, 7, 1709−1721. (56) Bratzel, G.; Buehler, M. J. Sequence-Structure Correlations in Silk: Poly-Ala Repeat of N. Clavipes MaSp1 Is Naturally Optimized at a Critical Length Scale. J. Mech. Behav. Biomed. Mater. 2012, 7, 30−40. (57) Patil, S. P.; Markert, B.; Gräter, F. Rate-Dependent Behavior of the Amorphous Phase of Spider Dragline Silk. Biophys. J. 2014, 106, 2511−2518. K
DOI: 10.1021/acs.macromol.8b00536 Macromolecules XXXX, XXX, XXX−XXX
Article
Macromolecules
Structure of Bombyx Mori Silk Fibroin. Macromolecules 1984, 17, 1075−1081. (79) Nagano, A.; Kikuchi, Y.; Sato, H.; Nakazawa, Y.; Asakura, T. Structural Characterization of Silk-Based Water-Soluble Peptides (Glu)n(Ala-Gly-Ser-Gly-Ala-Gly)4(n = 4−8) as a Mimic of Bombyx Mori Silk Fibroin by 13C Solid-State NMR. Macromolecules 2009, 42, 8950−8958. (80) Shoji, A.; Ozaki, T.; Fujito, T.; Deguchi, K.; Ando, I.; Magoshi, J. 15N Chemical Shift Tensors and Conformation of Solid Polypeptides Containing 15N-Labeled Glycine Residue by 15N NMR. J. Mol. Struct. 1998, 441, 251−266. (81) Asakura, T.; Yoshimizu, H.; Yoshizawa, F. NMR of Silk Fibroin 0.9. Sequence and Conformation Analyses of the Silk Fibroins from Bombyx Mori and Philosamia Cynthia Ricini by 15N NMR Spectroscopy. Macromolecules 1988, 21, 2038−2041. (82) Asakura, T.; Watanabe, Y.; Itoh, T. NMR of Silk Fibroin 0.3. Assignment of Carbonyl Carbon Resonances and Their Dependence on Sequence and Conformation in Bombyx Mori Silk Fibroin Using Selective Isotopic Labeling. Macromolecules 1984, 17, 2421−2426. (83) Saitô, H. Conformation-Dependent 13C Chemical Shifts: A New Means of Conformational Characterization as Obtained by HighResolution Solid-State 13C NMR. Magn. Reson. Chem. 1986, 24, 835− 852. (84) Saitô, H.; Ando, I. High-Resolution Solid-State NMR Studies of Synthetic and Biological Macromolecules. Annu. Rep. NMR Spectrosc. 1989, 21, 209−290. (85) Wishart, D. S. Interpreting Protein Chemical Shift Data. Prog. Nucl. Magn. Reson. Spectrosc. 2011, 58, 62−87. (86) Nicholson, L. K.; Asakura, T.; Demura, M.; Cross, T. A. A Method for Studying the Structure of Uniaxially Aligned Biopolymers Using Solid State 15N-Nmr: Application to Bombyx Mori Silk Fibroin Fibers. Biopolymers 1993, 33, 847−861. (87) Demura, M.; Yamazaki, Y.; Asakura, T.; Ogawa, K. Structure of Uniaxially Aligned 13C Labeled Silk Fibroin Fibers with Solid State 13 C-NMR. J. Mol. Struct. 1998, 441, 155−163. (88) Asakura, T.; Ito, T.; Okudaira, M.; Kameda, T. Structure of Alanine and Glycine Residues of Samia Cynthia Ricini Silk Fibers Studied with Solid-State 15N and 13C NMR. Macromolecules 1999, 32, 4940−4946. (89) Asakura, T.; Okushita, K.; Williamson, M. P. Analysis of the Structure of Bombyx mori Silk Fibroin by NMR. Macromolecules 2015, 48, 2345−2357.
(58) Ukpebor, O. T.; Shah, A.; Bazov, E.; Boutis, G. S.; Yarger, J. L.; Holland, G. P.; Callahan, D. J.; Zalutsky, M. R.; Chilkoti, A. Inverse Temperature Transition of Elastin like Motifs in Major Ampullate Dragline Silk: MD Simulations of Short Peptides and NMR Studies of Water Dynamics. Soft Matter 2014, 10, 773−785. (59) Giesa, T.; Perry, C. C.; Buehler, M. J. Secondary Structure Transition and Critical Stress for a Model of Spider Silk Assembly. Biomacromolecules 2016, 17, 427−436. (60) Sintya, E.; Alam, P. Self-Assembled Semi-Crystallinity at Parallel β-Sheet Nanocrystal Interfaces in Clustered MaSp1 (Spider Silk) Proteins. Mater. Sci. Eng., C 2016, 58, 366−371. (61) Giesa, T.; Schuetz, R.; Fratzl, P.; Buehler, M. J.; Masic, A. Unraveling the Molecular Requirements for Macroscopic Silk Supercontraction. ACS Nano 2017, 11, 9750−9758. (62) Plimpton, S. Fast Parallel Algorithms for Short-Range Molecular Dynamics. J. Comput. Phys. 1995, 117, 1−19. (63) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids. J. Am. Chem. Soc. 1996, 118, 11225−11236. (64) Asakura, T.; Nishimura, A.; Kametani, S.; Kawanishi, S.; Aoki, A.; Suzuki, F.; Kaji, H.; Naito, A. Refined Crystal Structure of Samia Cynthia Ricini Silk Fibroin Revealed by Solid-State NMR Investigations. Biomacromolecules 2017, 18, 1965−1974. (65) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79, 926−935. (66) Martínez, L.; Andrade, R.; Birgin, E. G.; Martínez, J. M. Packmol: A package for building initial configurations for molecular dynamics simulations. J. Comput. Chem. 2009, 30, 2157−2164. (67) Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23, 327−341. (68) Schneider, T.; Stoll, E. Molecular-dynamics study of a threedimensional one-component model for distortive phase transitions. Phys. Rev. B: Condens. Matter Mater. Phys. 1978, 17, 1302−1322. (69) Sugita, Y.; Okamoto, Y. Replica-Exchange Molecular Dynamics Method for Protein Folding. Chem. Phys. Lett. 1999, 314, 141−151. (70) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33−38. (71) Heyer, L. J.; Kruglyak, S.; Yooseph, S. Exploring Expression Data: Identification and Analysis of Coexpressed Genes. Genome Res. 1999, 9, 1106−1115. (72) Frishman, D.; Argos, P. Knowledge-Based Protein Secondary Structure Assignment. Proteins: Struct., Funct., Genet. 1995, 23, 566− 579. (73) Kabsch, W.; Sander, C. Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen-Bonded and Geometrical Features. Biopolymers 1983, 22, 2577−2637. (74) Asakura, T.; Iwadate, M.; Demura, M.; Williamson, M. P. Structural Analysis of Silk with 13C NMR Chemical Shift Contour Plots. Int. J. Biol. Macromol. 1999, 24, 167−171. (75) Asakura, T.; Ashida, J.; Yamane, T.; Kameda, T.; Nakazawa, Y.; Ohgo, K.; Komatsu, K. A Repeated β-Turn Structure in Poly(Ala-Gly) as a Model for Silk I of Bombyx Mori Silk Fibroin Studied with TwoDimensional Spin-Diffusion NMR under off Magic Angle Spinning and Rotational Echo Double Resonance. J. Mol. Biol. 2001, 306, 291− 305. (76) Asakura, T.; Yao, J.; Yamane, T.; Umemura, K.; Ulrich, A. S. Heterogeneous Structure of Silk Fibers from Bombyx Mori Resolved by 13 C Solid-State NMR Spectroscopy. J. Am. Chem. Soc. 2002, 124, 8794−8795. (77) Asakura, T.; Yao, J. 13C CP/MAS NMR Study on Structural Heterogeneity in Bombyx Mori Silk Fiber and Their Generation by Stretching. Protein Sci. 2002, 11, 2706−2713. (78) Asakura, T.; Watanabe, Y.; Uchida, A.; Minagawa, H. NMR of Silk fibroin.2. 13C NMR Study of the Chain Dynamics and Solution L
DOI: 10.1021/acs.macromol.8b00536 Macromolecules XXXX, XXX, XXX−XXX
