Dynamics of Alanine Methyl Groups in Alanine Oligopeptides and

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

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Dynamics of Alanine Methyl Groups in Alanine Oligopeptides and Spider Dragline Silks with Different Packing Structures As Studied by 13C Solid-State NMR Relaxation Tetsuo Asakura,* Yugo Tasei, Hironori Matsuda, and Akira Naito Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan

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S Supporting Information *

ABSTRACT: The dynamics of alanine methyl groups in alanine oligopeptides and polyalanine regions in Nephila clavata dragline and recombinant silks changed remarkably depending on the different packing arrangements. In particular, a notable difference in the 13C spin−lattice relaxation time T1 of Ala Cβ peaks was observed in the solid-state NMR between (Ala)6 and (Ala)7, which have rectangular and staggered packing arrangements, respectively. Moreover, the remarkably longer T1 value of the lowest field peak in Ala Cβ carbons clearly indicated the presence of staggered packing structure. Then, the relaxation behavior of polyalanine regions in spider dragline silks with mixed packing arrangements were clarified in both dry and hydrated states. The partial appearance of the staggered packing structure in (Ala)6 with rectangular packing structure was detected from the changes in line shapes of Ala 13Cβ peaks for the three kinds of 13C single-labeled (Ala)6 samples together with the change in their relaxation behavior.

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strength, stiffness, and toughness than larger β-crystallites. In addition, the relationship between the crystallite orientation and breaking stress of silkworm and spider dragline silk fibers has been studied by using theoretical16 and experimental methods.14,17 Typically, a higher orientation of β-crystallites along fiber axis contributed to a higher breaking stress. Thus, the excellent mechanical properties of spider dragline silk fibers can be interpreted as due to the size and orientation of βcrystallites. However, there has been very little information about the packing structure of poly-A region with AP-β structure, and theoretical approaches such as MD simulation and molecular mechanics calculations of spider dragline silk fiber need to be conducted with accurate packing structures for AP-β poly-A regions in appropriate β-crystallite models. In our previous papers,18−20 two kinds of packing arrangements depending on the number of Ala residue could be discerned from experimental 13C solid-state NMR data. A rectangular arrangement was reported for (Ala)6 and shorter poly-A chains, while a staggered arrangement was found for (Ala)7 and longer poly-A chains. In addition, we determined the packing arrangements of a wild silkworm, Samia cynthia ricini (S. c. ricini) silk fibroin, which had a similar primary structure as spider dragline silk, i.e., block copolymers with alternating poly-A and Gly-rich blocks.21 The main difference between the silk fibroin and the spider silk was a longer poly-A sequence in the silk fibroin, i.e., (Ala)12−13, relative to (Ala)n (n

INTRODUCTION Spider dragline silk fibers have been of great interest because of their excellent mechanical properties, i.e., high strength and high toughness, leading to many possibilities of their applications in diverse fields.1−4 These properties can be attributed to the protein’s conformation and packing structures, which arise from their unique primary structure, comprising block copolymers with alternating polyalanine (poly-A) and glycine-rich blocks.5,6 In general, the former polyA region has been associated with high fiber strength, and the latter glycine-rich region is a source of the high elasticity. The conformation of poly-A region has been confirmed to contain antiparallel β-sheet (AP-β) structure and β-crystallites.2,3 In βcrystallites, there are intermolecular hydrogen bonds, together with intersheet van der Waals and hydrophobic interactions, that contribute significantly to the stability of the structure. The size of β-crystallites of Naphila clavipes (N. clavipes) dragline silk fiber has been studied using both XRD and TEM.3,7−13 Grubb and Jelinski7 reported that the size was very small and well-oriented. Thus, the mean (minimum) crystal dimensions were 2 × 5 × 7 nm3, and the angle between the molecular chains in the crystals and the fiber axis had a full width at half-maximum of 15.7°. Other reports8−14 basically supported their results. Keten et al.15 performed a series of large-scale molecular dynamics (MD) simulations to explore the correlation between the size effect of β-crystallite and the mechanical property of silk fibroin and indicated that the mechanical properties of silk are strongly dependent on the βsheet crystallite sizes. Thus, they reported that β-crystallites are confined to a few nanometers which can impart higher © XXXX American Chemical Society

Received: July 1, 2018 Revised: August 10, 2018

A

DOI: 10.1021/acs.macromol.8b01402 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules = 6 or 7) in the spider silk.5,6,21 The poly-A sequences in S. c. ricini silk fiber were packed in the staggered arrangement, and the atomic-level structure was determined.22,23 On the basis of these studies, for the first time we reported that the packing arrangements of AP-β poly-A regions in Nephila clavata (N. clavata) dragline silk fiber (NCF) and recombinant dragline silk protein (RSP) with the sequences from ADF-3 silk protein from the European garden spider Araneus diadematus (A. diadematus) were mixtures of the rectangular and staggered packing arrangements.20 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 were determined to be 49% and 51%, respectively, in the dry state and 40% and 60% in the hydrated state for AP-β NCF and were 62% and 38% in the dry state and 81% and 19% in the hydrated state for AP-β RSP. Thus, the fraction of packing structures changed significantly between the two spider silks and also between the two physical states. This is very interesting because the length of poly-A sequences in spider dragline silk fibers is mostly 6 or 7.5,6 Another important factor that impacts the mechanical properties is the dynamics of amino acid residues in spider dragline silk fiber although it has been discussed previously in connection with the domain structure. Solid-state NMR has the advantage that it can provide detailed structural information and can also give the dynamical information at different motional levels from the side-chain fast motion to the slower motion of the backbone chain of the spider dragline silks.24,25 Indeed, many NMR papers on this topic have been published including ours.26−39 Simmons et al.26 reported the presence of two components with significantly different mobilities for Ala residues in the crystalline poly-A regions of N. clavipes dragline silk fiber from the 13C CP/MAS NMR relaxation studies. They also reported two types of poly-A sequences: one that is highly oriented and another that is poorly oriented and less densely packed from the line shape analysis of the 2H solid-state NMR spectrum of [3,3,3-2H3]AlaN. clavipes dragline silk fiber.27 Shi et al.31 also reported two components for [3,3,3-2H3]Ala-N. clavipes dragline silk fiber with 2H spin−lattice relaxation times (T1’s) measured indirectly using 13C detected 2H−13C CP/MAS NMR in the dry and hydrated states. Most recently, we studied the dynamics and rectangular packing arrangements of Ala oligopeptides, (Ala) 3 and (Ala) 4 , with known atomic coordinate data from 13C solid-state T1 observations.40 The T1 values of the Ala Cβ carbons in the solid state reflect the anisotropic hopping motion of the methyl groups, which is reflected in the correlation times (τc’s). The τc values can be evaluated from the T1 values together with temperature dependence of T1 values with the help of an equation expressing T1 values of methyl carbons that takes into account the anisotropic hopping motions of the methyl group in the solid state. This is based on the fact that the methyl hopping motion is well characterized and dominates the 13C spin− lattice relaxation mechanism of Ala Cβ carbons of poly-A region in the τc values of 10−9−10−11 s.31,41,42 We also observed 13C T1 values of Ala Cβ peaks in the 13C CP/MAS NMR spectra of S. c. ricini SF fibers to clarify the dynamical behavior of Ala residues in the dry and hydrated states. Unusual dynamic character was observed in Ala Cβ carbon in the poly-A region of silk fibroin fiber of S. c. ricini in staggered packing structure from 13C solid-state T1 observations.43

In this paper, we focused on the 13C solid-state T1 determinations of Ala Cβ carbons in a series of Ala oligopeptides ((Ala)n where n = 5, 6, 7, and 12) and two kinds of spider dragline silks, NCF and RSP.44,45 First, we clarified the remarkable difference in the relaxation behavior of Ala residues with AP-β structure among several alanine oligopeptides, some shorter than (Ala)6 and some longer than (Ala)7, with rectangular and staggered packing arrangements, respectively. Second, the relaxation behavior of Ala residues in [3-13C]Ala-NCF and [3-13C]Ala-RSP was discussed in relation to the difference in the packing structures between two spider silks in the dry and hydrated states. From the temperature dependencs of 13C T1 values of Ala Cβ carbons in both silks, the τc values were calculated. Finally, the partial appearance of staggered packing structure in (Ala)6 with rectangular packing structure was detected from the characteristic shapes of Ala 13Cβ peaks of three kinds of 13C singlelabeled (Ala)6 samples, Ala[3-13C]Ala2(Ala)4, (Ala)2[3-13C]Ala3(Ala)3, and (Ala)4[3-13C]Ala5Ala, together with their relaxation behavior.

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MATERIALS AND METHODS

Preparation of Alanine Oligopeptides. (Ala)n (n = 5 and 6) were purchased from Bachen AG (Bunderdorf, Switzerland) and used without further purification. Other peptides, (Ala)n (n = 7 and 12), and three kinds of 13C single-labeled (Ala)6 samples, Ala[3-13C]Ala2(Ala)4, (Ala)2[3-13C]Ala3(Ala)3, and (Ala)4[3-13C]Ala5Ala, used here were synthesized by the Fmoc solid phase method (Pioneer Peptide Synthesizer, PE. Biosystems Co., Ltd.) in the Asakura laboratory. Details of the peptide synthesis were described elsewhere.19 (Ala)5 and (Ala)6 were dissolved in aqueous solution and then lyophilized. (Ala)7 and (Ala)12 were dissolved in 55% (w/w) lithium thiocyanate and then dialyzed against water for 3 days and finally collected by lyophilization.19 The AP-β structures of these peptides were confirmed by 13C solid-state NMR and FT-IR methods.18−20 The packing arrangements of these samples were also identified from the patterns of the Ala Cβ peaks. [3-13C]Ala (each 99% enrichment) was purchased from Cambridge Isotope Laboratories, Inc., Andover, MA. [3-13C]Ala-N. clavata Dragline Silk Fiber and [3-13C]AlaRecombinant Dragline Silk. The spiders (N. clavata) were taken at the campus of the Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan, and the dragline silk fibers were collected from them. The [3-13C]Ala-NCF was performed by giving droplet of aqueous solution of [3-13C]Ala (10% w/v) to the spiders.18,20 The [3-13C]Ala-RSP powder with the sequence as the ADF-3 silk protein from the European garden spider A. diadematus was prepared as described previously.45 The powder sample was dried after immersing it in methanol to convert the conformation to AP-β structure.45 13 C Solid-State NMR Measurements. 13C solid-state NMR spectra of alanine oligopeptides (Ala)n (n = 5, 6, 7, and 12) in the dry state were recorded on a Chemagnetics Infinity 400 NMR spectrometer with MAS frequency of 7 kHz using a Chemagnetics 4 mm HXY T-3 MAS probe head at 25 °C. Furthermore, 13C solidstate NMR spectra of three kinds of 13C single-labeled (Ala)6 samples in dry state were recorded on Bruker Avance 400 NMR spectrometers at MAS frequency of 10 kHz using double resonance MAS probe and a rotor with 4 mm outer diameter at 25 °C. Typical experimental parameters for the 13C CP/MAS NMR observations included 3.5 μs 1 H 90° pulse, 2 ms ramped CP pulse with average 70 kHz rf field strength, TPPM 1H decoupling during acquisition, 2048 data points, and 5 s recycle delay. The 13C T1’s were determined for Ala Cβ carbons of all samples by the method of Torchia.46 The temperaturedependent 13C T1 experiments observed at 25, 40, and 60 °C were performed for [3-13C]Ala-NCF and [3-13C]Ala-RSP samples in dry and hydrated states using Bruker Avance 400 NMR spectrometers at the same experimental condition. For the T1 observations of hydrated B


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Figure 1. A series of partially relaxed 13C CP/MAS NMR spectra of Ala Cβ peaks of (a) (Ala)5, (b) (Ala)6, (c) (Ala)7, and (d) (Ala)12 in the dry state at 25 °C as a function of delay time τ for T1 determinations. The range of τ values was from 10 to 500 ms for (a) and (b) and from 10 to 3000 ms for (c) and (d).

Table 1. 13C Solid-State Spin−Lattice Relaxation Times (T1’s, in s) and 13C Chemical Shifts [in ppm from TMS] of Ala Cβ Peaks of a Series of AP-β Ala Oligopeptides, (Ala)n (n = 5, 6, 7, and 12), S. c. Ricini Silk Fiber,a and Three Kinds of 13CLabeled-(Ala)6, i.e., Ala[3-13C]Ala2(Ala)4, (Ala)2[3-13C]Ala3(Ala)3, and (Ala)4[3-13C]Ala5Ala in the Dry State at 25 °Cb T1 (s) [13C chemical shift (ppm)]

samples (Ala)5 (Ala)6 (Ala)7 (Ala)12 S. c. ricini silk fibera Ala[3-13C]Ala2(Ala)4 (Ala)2[3-13C]Ala3(Ala)3 (Ala)4[3-13C]Ala5Ala

0.15 [21.2] ± 0.01 0.95 1.33 1.01 0.52 1.38 0.50

[22.7] [22.7] [22.7] [22.0] [22.7] [22.0]

± ± ± ± ± ±

0.14 0.05 0.03 0.04 0.06 0.03

0.13 [20.5] ± 0.01 0.16 [20.6] ± 0.01

0.11 [19.1] ± 0.01 0.33 [19.6] ± 0.01 0.43 [19.6] ± 0.02 0.55 [19.6] ± 0.02

0.62 [21.2] ± 0.02 0.24 [20.6] ± 0.01 0.17 [20.6] ± 0.01 0.24 [20.3] ± 0.01

0.28 [21.2] ± 0.01

0.62 [16.6] ± 0.02

0.38 [19.6] ± 0.01

Reference 43. bThe error ranges are obtained as standard deviation of the relaxation times in the fitting processes.The assignment of each peak is shown in the text. a

[3-13C]Ala-NCF and hydrated [3-13C]Ala-RSP, the samples were inserted into a zirconia rotor that was sealed with a PTFE insert to prevent dehydration of the hydrated samples.20,45 The chemical shifts were referenced to TMS, using adamantine as an external standard (13CH peak at 28.8 ppm). The 13C T1 values were determined by using changes in the intensities of the peaks with the fixed chemical shifts of a partially relaxed spectra. The standard deviations of the T1 values were calculated in the fitting process. Analysis of 13C Solid-State T1 Value of Ala Cβ Peak. The 13C solid-state T1 values for Ala Cβ carbons with hopping motion under the solid-state MAS condition can be expressed as41,47

γC2γH 2ℏ2I(I + 1) ÄÅÅÅ 1 (0) 1 ÅÅ J (ωC − ωH) + 3 J (1)(ωC) = ÅÅ 12 2 T1C r6 ÅÇ ÉÑ ÑÑ 3 (2) + J (ωC + ωH)ÑÑÑ ÑÑÖ 4

J (0)(ω) =

ÅÄÅ ÑÉÑ Å ÑÑ τc 6 ÑÑ (sin 2 2Δ + sin 4 Δ)ÅÅÅÅ ÅÅ 1 + ω2τc 2 ÑÑÑ 5 ÅÇ ÑÖ

ÄÅ ÉÑ ÅÅ ÑÑ τc 1 Å ÑÑ 2 4 Å J (ω) = (sin 2Δ + sin Δ)ÅÅ Ñ 2 2Ñ Å 5 ÅÅÇ 1 + ω τc ÑÑÑÖ (1)

ÄÅ ÉÑ ÅÅ ÑÑ τc 4 Å ÑÑ 2 4 Å J (ω) = (sin 2Δ + sin Δ)ÅÅ Ñ 2 2 ÅÅ 1 + ω τc ÑÑÑ 5 ÅÇ ÑÖ (2)

Here, I is the spin quantum number of the proton (1/2), and ωC and ωH are Larmor angular frequencies of carbon and proton nuclei, respectively. Δ is the angle between the C−H internuclear vector and the C3 axis, and τc is the correlation time of the hopping motion of methyl groups about the C3 axis. Through the use of eq 1 and parameters of the anisotropic motion of methyl group (Δ = 69.8°, rCH = 1.098 Å cited from neutron scattering study of L-alanine crystal48), a plot of T1 values versus correlation times could be calculated

(1)

where C


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(Supporting Information Figure S1). The calculated result indicates a T1 minimum value of 0.0602 s with τc of 1.26 × 10−9 s at the frequency of 126 MHz in the magnetic field of 9.4 T. (Larmor frequencies of carbon and proton nuclei are 100 and 400 MHz, respectively.) Once the temperature dependence of the T1 value is measured, the τc of the Cβ hopping motion can be determined using eq 1, which was also applied hereinafter for [3-13C]Ala-NCF and [3-13C]Ala-RSP samples.

C Spin−Lattice Relaxation Times of Ala Cβ Carbons in Spider Silks, [3-13C]Ala-NCF and [3-13C]Ala-RSP in Dry and Hydrated States. Next, 13C T1 values of Ala Cβ carbons in [3-13 C]Ala-NCF and [3-13C]Ala-RSP samples were determined. In these samples, Ala Cβ carbons in AP-β polyA region with different packing structures can be selectively observed using CP/MAS experiment as compared with DD/ MAS and INEPT experiments as shown in the Supporting Information (Figures S2 and S3).20,45 Thus, 13C CP/MAS experiments were performed to determine the T1 values for NCF and RSP samples. As shown in Figure 2a, the expanded

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RESULTS C Spin−Lattice Relaxation Times of Ala Cβ Carbons in Alanine Oligopeptides ((Ala)n (n = 5, 6, 7, and 12)) in the Dry State. A series of partially relaxed 13C CP/MAS NMR spectra of Ala Cβ peaks of AP-β (Ala)n (n = 5, 6, 7, and 12) in the dry state are shown in Figure 1 for the T1 determinations at 25 °C. The 13C T1 values are summarized in Table 1. In our previous papers,18−20 we assigned the Ala Cβ peaks of these alanine oligopeptides to the conformations and the packing arrangements of AP-β structure. In this paper, we will clarify the difference in the dynamical behaviors of these Ala Cβ peaks between the rectangular groups ((Ala)5,6) and staggered groups ((Ala)7,12). The packing arrangement of (Ala)5 was reported to be rectangular previously.18 As summarized in Table 1, there is no significant difference in the T1 values (0.11−0.15 s) among three Ala Cβ peaks of (Ala)5. In (Ala)6, the 13C CP/MAS spectrum apparently comprises a single peak, and the chemical shift is essentially the same as that of the strongest peak of (Ala)5. The T1 value (0.16 s) of Ala Cβ peak was also almost the same as those of (Ala)5. Thus, the packing structure of (Ala)6 is considered to be rectangular like (Ala)5 from the viewpoint of the T1 observation, too.18 However, the structural and dynamical behavior of the individual residue in (Ala)6 is complex as will be described later. In contrast, the line shape of Ala Cβ carbons of (Ala)7 was quite different from that of (Ala)6, which reflects the change in the packing structure from rectangular to staggered.18−20 Thus, it is of interest to study the change in the dynamical behavior due to the change in the packing structure. The T1 value 0.33 s of the highest field peak at 19.6 ppm for the Ala Cβ carbon of (Ala)7 was 2 times longer than the T1 value 0.16 s of the single peak at 20.6 ppm of (Ala)6. Moreover, the T1 value 0.95 s of the newly observed Ala Cβ peak at 22.7 ppm was 3 times longer than that of 19.6 ppm peak of (Ala)7. The T1 values of Ala Cβ carbons of (Ala)12 have a similar trend as that of (Ala)7, but the absolute T1 values became slightly longer; namely, the T1 value of the 19.6 ppm peak was 0.43 s, and that of the 22.7 ppm peak was 1.33 s. It is important to point out that the two peaks at 22.7 and 19.6 ppm in (Ala)7 and (Ala)12 belong to the staggered packing arrangement reported previously.18 Thus, the observed T1 values of Ala Cβ peaks of AP-β (Ala)n (n = 5, 6, 7, and 12) also reflect the change in the packing arrangement from rectangular to staggered as well as the change in the line shapes. The remarkably longer T1 value, 1.01 s, of 22.7 ppm than that of 19.6 ppm, 0.55 s, was also reported for S. c. ricini silk fibroin fiber with the staggered packing arrangement previously (Table 1).43,44 The extremely long T1 value of the 22.7 ppm peak was interpreted in terms of a geared hopping motion of Ala Cβ carbons in poly-A region of S. c. ricini silk fibroin fiber through a combination of 13C solid-state NMR relaxation time measurement and MD simulation. 13

Figure 2. Deconvolution of Ala Cβ peaks in the 13C CP/MAS NMR spectra of two kinds of spider silks in the dry state at 25 °C: (a) AP-β [3-13C]Ala-NCF and (b) AP-β [3-13C]Ala-RSP reported previously.20 Spectrum (a) was deconvoluted into three components corresponding to AP-β signals with rectangular arrangement (purple broken line), AP-β spectrum with staggered arrangement (blue broken line), and random coil (orange broken line) with the fractions of 38%, 39%, and 23%, respectively.Spectrum (b) was also deconvoluted into three components similarly, but an additional peak of Pro Cγ carbon was involved. Except for the additional peak, the fraction was 47% (rectangular), 28% (staggered), and 25% (random coil). Purple broken lines: Ala Cβ resonances of (Ala)6; light blue broken lines: Ala Cβ resonances of (Ala)7 resonances; orange broken lines: Ala Cβ random coil resonances; green broken lines: Pro Cγ resonance.

Ala Cβ peak of AP-β [3-13C]Ala-NCF sample in the dray state at 25 °C was deconvoluted into three components, i.e., random coil and two kinds of packing arrangement of the AP-β poly-A sequence, (Ala)6 (rectangular packing) and (Ala)7 (staggered packing).20 The component fractions of two AP-β sheet resonances were 38% for rectangular, 39% for staggered packing arrangements, and 23% random coil. On the other hand, in the 13C CP/MAS NMR spectrum of AP-β [3-13C]AlaRSP powder sample after methanol treatment in the dry state at 25 °C (Figure 2b), the Ala Cβ resonance region was deconvoluted into four peaks.45 Namely, one additional peak of Pro Cγ carbon at 24.6 ppm was observed other than the three Ala Cβ peaks. Three components of Ala Cβ peak corresponded to 47% for rectangular and 28% for staggered packings of the AP-β poly-A sequence and 25% for the random coil. Figure 3 shows a series of partially relaxed 13C CP/MAS NMR spectra of the Ala Cβ peaks of Ap-β [3-13C]Ala-NCF and AP-β [3-13C]Ala-RSP samples in the dry state at 25 °C. In Figure 3a, it is difficult to separate the rectangular peak from the staggered peak in the T1 determination because of the severe overlap of two heterogeneous peaks with different T1 values. Thus, the T1 values were determined by fixing the chemical shifts of three peaks: 16.6, 20.2, and 22.9 ppm as listed in Table 2. The plots of log Mτ vs τ values of individual peaks in Figures 3 were summarized in the Supporting Information (Figure S4) together with those in Figure 4 and D


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state at 25 °C were observed as shown in Figure 3b. Here, to deduct the contribution of the Pro Cγ peak at 24.6 ppm in Figure 2b to the spectra, the difference spectra ([3-13C]AlaRSP-nonlabeled Ala-RSP) were obtained (Figure S5). The central main peak was observed at 20.6 ppm (rectangular packing peak) instead of 20.2 ppm in the [3-13C]Ala-NCF sample. The T1 value was 0.29 s, which was almost one-half of the T1 value of the main peak at 20.2 ppm of [3-13C]Ala-NCF as listed in Table 2. This tendency is similar to the case of main peaks of alanine oligopeptides, (Ala)6 and (Ala)7, as listed in Table 1 although absolute T1 values were different between alanine oligopeptides and spider dragline silks. In particular, the T1 value of the highest field peak at 19.6 ppm was 2 times longer than that of the main peak at 20.6 ppm of (Ala)6. The T1 values of the other peaks [16.6 ppm (random coil) and 22.9 ppm (lower field: staggered peak)] were almost the same between [3-13C]Ala-NCF and [3-13C]Ala-RSP. Thus, the lowest field peak at 22.9 ppm has the longest T1 value among all peaks, indicating the presence of the staggered arrangement in the sample. This result is partly similar to what was found in [3-13C]Ala-NCF. In contrast, a series of partially relaxed 13C CP/MAS NMR spectra of the Ala Cβ peaks of AP-β [3-13C]Ala-NCF and AP-β [3-13C]Ala-RSP samples in the hydrated state at 25 °C are shown in Figure 4. Both [3-13C]Ala-NCF and [3-13C]Ala-RSP spectra tend to be sharper than those in the dry state. This is due to a significant loss in the CP signals of some of Ala Cβ carbons because of increased mobility by hydration.20,28,31,45 In particular, some part of random coil region disappeared in the 13 C CP/MAS NMR spectra of AP-β [3-13C]Ala-NCF (Figure 4a), and the random coil peak disappeared significantly in those of AP-β [3-13C]Ala-RSP (Figure 4b). Such spectral changes were previously reported as shown in the Supporting Information (Figure S2 for [3-13C]Ala-NCF20 and Figure S3 for [3-13C]Ala-RSP45). In addition, because of hydration, all of the observed peaks became slightly sharper, and as a result, the lowest field β-sheet peak at 22.9 ppm could be observed clearly in [3-13C]Ala-NCF. All of the T1 values of [3-13C]Ala-NCF

Figure 3. A series of partially relaxed 13C CP/MAS NMR spectra of Ala Cβ peaks of (a) AP-β [3-13C]Ala-NCF and (b) AP-β [3-13C]AlaRSP in the dry state at 25 °C as a function of delay time τ for T1 determinations. In spectra (a), the assignment is 16.6 ppm (random coil), 20.2 ppm (mixture of rectangular and staggered arrangements), and 22.9 ppm (staggered arrangement). In spectra, (b), the peak of Pro Cγ carbon at 24.6 ppm in Figure 2b was deleted by subtraction of nonlabeled RSP spectrum from the [3-13C]Ala-RSP spectrum. The assignment is 16.6 ppm (random coil), 20.6 ppm (rectangular arrangement), and 22.9 ppm (staggered arrangement). The vertical guide lines (broken lines) were added to the spectra.

Table 2. Here Mτ means the peak intensity at τ s. The plots could be considered to be linear although we used the peak heights instead of the peak area. The T1 values were 0.61 s for 16.6 ppm (random coil), 0.57 s for β-sheet (main peak at 20.2 ppm: mixture of rectangular and staggered peaks), and 0.93 s for β-sheet (22.9 ppm: lower field staggered peak). These T1 values were close to the values, i.e., 0.62 s for 16.6 ppm (random coil), 0.55 s for β-sheet (higher field staggered peak), and 1.01 s for β-sheet (22.7 ppm: lower field staggered peak), reported for Ala Cβ peaks of S. c. ricini silk fibroin fiber with the staggered packing arrangement43 as listed in Table 1. Thus, in the T1 determination of the main Ala Cβ peak at 20.2 ppm of Ap-β [3-13C]Ala-NCF, the contribution of the rectangular packing structure in the sample is relatively small. In contrast, a series of partially relaxed 13C CP/MAS NMR spectra of the Ala Cβ peaks of AP-β [3-13C]Ala-RSP in the dry

Table 2. 13C Solid-State Spin−Lattice Relaxation Times (T1’s, in s) and 13C Chemical Shifts [CS, in ppm from TMS] of Ala Cβ Peaks of AP-β [3-13C]Ala-NCF and AP-β [3-13C]Ala-RSP in the Dry and Hydrated States: (a) Dry NCF, (b) Dry RSP, (c) Hydrated NCF, and (d) Hydrated RSP at Temperature 25, 40, and 60 °Ca (a) dry NCF

(c) hydrated NCF

CS

25 °C

40 °C

60 °C

CS

[22.9]

0.93 ± 0.02

0.90 ± 0.05

0.92 ± 0.02

[22.9]

[20.2]

0.57 ± 0.02

0.56 ± 0.02

0.58 ± 0.02

[20.2]

[16.6]

0.61 ± 0.02

0.69 ± 0.02

0.66 ± 0.02

[17.2]

(b) dry RSP

25 °C

40 °C

0.88 ± 0.02 0.92 ± 0.01 2.04 1.95 0.44 ± 0.02 0.50 ± 0.02 4.11 3.60 0.48 ± 0.01 0.52 ± 0.01 3.76 3.46 (d) hydrated RSP

60 °C 0.96 ± 0.01 1.87 0.53 ± 0.02 3.40 0.54 ± 0.01 3.33

CS

25 °C

40 °C

60 °C

CS

25 °C

40 °C

60 °C

[22.9]

0.84 ± 0.14

0.85 ± 0.08

0.76 ± 0.11

[22.9]

[20.6]

0.29 ± 0.01

0.23 ± 0.01

0.21 ± 0.01

[20.6]

0.81 ± 0.08 2.21 0.23 ± 0.01 8.09

0.90 ± 0.12 1.99 0.25 ± 0.01 7.40

0.96 ± 0.05 1.87 0.27 ± 0.01 6.81

[16.6]

0.61 ± 0.05

0.57 ± 0.15

0.48 ± 0.04

a The plots of log Mτ vs τ values of individual peaks are shown in Figure S4. The error ranges are obtained as standard deviation of the relaxation times in the fitting processes the correlation times, τc (in ×10−11 s), listed under T1 values are calculated using eq 1 in the hydrated state.

E


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Figure 4. A series of partially relaxed 13C CP/MAS NMR spectra of Ala Cβ peaks of (a) AP-β [3-13C]Ala-NCF and (b) AP-β [3-13C]Ala-RSP in the hydrated state at 25 °C as a function of delay time τ for T1 determinations. In spectra, (a), the assignment is 17.2 ppm (random coil), 20.2 ppm (mixture of rectangular and staggered arrangements), and 22.9 ppm (staggered arrangement). In spectra, (b), the peak of Pro Cγ carbon at 24.6 ppm in Figure 2b was deleted by subtraction of nonlabeled RSP spectrum from the [3-13C]Ala-RSP spectrum. The assignment is 20.6 ppm (rectangular arrangement) and 22.9 ppm (staggered arrangement). The vertical guide lines (broken lines) were added to the spectra.

Figure 5. Plots of 13C spin−lattice relaxation time values (T1’s, in s) of Ala Cβ peaks of (a) AP-β [3-13C]Ala-NCF and (b) AP-β [3-13C]Ala-RSP in the hydrated state against reciprocal absolute temperatures (1/T). The error bars were included. The temperature dependence of the T1 value is used to uniquely determine τc value of the hopping motion using eq 1.

and [3-13C]Ala-RSP in the hydrated state became slightly smaller compared with those in the dry state (Table 2). Temperature Dependence of 13C Spin−Lattice Relaxation Times of Ala Cβ Carbons in Spider Silks and [3-13C]Ala-NCF and [3-13C]Ala-RSP in the Dry and Hydrated States. The 13C T1 value for Ala Cβ carbon is quantitatively characterized using eq 1 in which 1H3-13C hopping motion of MAS condition is considered. A plot of T1 values against τc is calculated using eq 1 as shown in Figure S1. The τc value is known to be proportional to 1/T, where T is the absolute temperature in units of kelvin. Once the temperature dependence of the T1 value is measured against the reciprocal temperature, it is possible to uniquely determine τc of the hopping motion using eq 1. The temperature dependences of T1 values were observed in dry and hydrated states as listed in Table 2. For AP-β [3-13C]Ala-NCF in the dry state, there are no significant change with increasing temperature within experimental error. For AP-β [3-13C]Ala-RSP in the dry state, the T1 values tend to decrease with increasing temperature, but the experimental errors in the T1 determination are relatively large. These results might come from the components of different groups with relatively broad relaxation

rates. However, because of hydration, the observed peaks of AP-β [3-13C]Ala-NCF and AP-β [3-13C]Ala-RSP became relatively sharper because of the loss of relatively fast motional components. As a result, the T1 values increased slightly with increasing temperature for both [3-13 C]Ala-NCF and [3-13C]Ala-RSP samples as reported previously for S. c. ricini silk fibroin fiber.43 Therefore, T1 values of both spider silk samples determined for only the hydrated states were plotted against reciprocal temperature (1/T) as summarized in Figure 5. The T1 values increased at higher temperatures (lower reciprocal temperatures), which are located at the position in the shorter τc region at the T1 minimum in the plot of T1 values against the 1/T (k−1) values. Based on this relationship, the τc values of the hopping motion of Ala Cβ groups in both spider silks in the hydrated state were uniquely determined using eq 1 as summarized in Table 2. Thus, the motional behavior of the lowest field peak shows the fastest hopping motion of the Cβ carbon about the C3 axis of Ala Cβ group in hydrated state for both AP-β [3-13C]Ala-NCF and AP-β [3-13C]Ala-RSP. 13 C CP/MAS NMR Spectra and 13C Spin−Lattice Relaxation Times of Different Ala 13Cβ Carbons in F


Article

Macromolecules Ala[3- 13 C]Ala 2 (Ala) 4 , (Ala) 2 [3- 13 C]Ala 3 (Ala) 3 , and (Ala)4[3-13C]Ala5Ala in the Dry State. The packing arrangement of AP-β (Ala)6 is considered to be rectangular like AP-β (Ala)5. However, the spectral pattern consisted of an apparent single peak with no clear peak splitting for AP-β (Ala)6 and is different from the spectrum of AP-β (Ala)5. As reported previously,19 the change in the packing pattern from rectangular to staggered arrangements was observed for AP-β (Ala)6 by heating the sample at 200 °C for 4 h. This high sensitivity of change in the packing structure of AP-β (Ala)6 due to a change in the environment around the molecules is in contrast with the case of more stable AP-β (Ala)7 with the staggered packing structure.19,20,23 Thus, the line shape and dynamics of individual residue in AP-β (Ala)6 were examined carefully by preparing AP-β (Ala)6 samples with different 13Cβ labeling positions. Figure 6 shows 13C CP/MAS NMR spectra of the Ala Cβ peaks of (a) nonlabeled AP-β (Ala)6, (b) nonlabeled AP-β

residue reflects the heterogeneous environment of AP-β (Ala)6, and the staggered packing is partly found. These data indicate that the packing structure changes depending on the position of the specified 13C carbon located in AP-β (Ala)6. Therefore, to evaluate the fractions of rectangular and staggered packing arrangements for the individual Ala 13Cβ carbons, the line shapes were calculated by changing the fraction of nonlabeled AP-β (Ala)6 and nonlabeled AP-β (Ala)7 patterns systematically as shown in Figure 7. The relative ratio of nonlabeled AP-

Figure 7. Ala Cβ line shapes calculated by changing the fraction of the lines shapes of Ala Cβ carbons of nonlabeled AP-β (Ala)6 and nonlabeled AP-β (Ala)7 systematically.

β (Ala)6 and nonlabeled AP-β (Ala)7 seems to be roughly 8:2 for Ala 13Cβ peak (Figure 6c) of AP-β Ala[3-13C]Ala2(Ala)4. Similarly, the relative ratio seems to be roughly 6:4 for Ala 13 Cβ peak (Figure 6d) of AP-β (Ala)2[3-13C]Ala3(Ala)3 although the central peak assigned to nonlabeled AP-β (Ala)6 became slightly sharper in peak (d). Nonetheless, it seems difficult to reproduce the Ala 13Cβ peak (Figure 6e) of AP-β (Ala)4[3-13C]Ala5Ala. Indeed, two main peaks with the chemical shifts of 20.3 and 21.2 ppm were observed. However, two higher field peaks occur at 19.6 and 20.6 ppm in the calculated 4:6 spectrum in Figure 7. In addition, the relative intensity of the lowest field at 22.7 ppm in peak (e) is considerably smaller than the calculated 4:6 peak although the presence of the peak at 22.7 ppm was observed clearly in the partially relaxed spectrum as shown below. These discrepancies might come from the generation of incomplete packing structures instead of distinct rectangular and staggered packing ones, especially the possibility of the disturbance of AP-β structure at the C-terminal area of linear (Ala)6 molecule. Figure 8 shows a series of partially relaxed 13C CP/MAS NMR spectra of the Ala Cβ peaks of (a) AP-β Ala[3-13C]Ala2(Ala)4, (b) AP-β (Ala)2[3-13C]Ala3(Ala)3, and (c) AP-β (Ala)4[3-13C]Ala5Ala in the dry state at 25 °C. The T1 value of

Figure 6. 13C CP/MAS NMR spectra of the Ala Cβ peaks of (a) nonlabeled AP-β (Ala)6 with rectangular arrangement and (b) nonlabeled AP-β (Ala)7 with staggered arrangement in the dry state at 25 °C. Those of the Ala13β peaks of 13C single-labeled AP-β (Ala)6 samples, i.e., (c) Ala[3-13C]Ala2(Ala)4, (d) (Ala)2[3-13C]Ala3(Ala)3, and (e) (Ala)4[3-13C]Ala5Ala, are also shown.

(Ala) 7 , (c) AP-β Ala[3- 1 3 C]Ala 2 (Ala) 4 , (d) AP-β (Ala)2[3-13C]Ala3(Ala)3, and (e) AP-β (Ala)4[3-13C]Ala5Ala in the dry state at 25 °C. It is noted that the shape of Ala 13Cβ peak changes remarkably depending on the13C-labeled position. More specifically, the Ala 13C β spectral region of AP-β Ala[3-13C]Ala2(Ala)4 seems to show a single peak, which is similar to that of nonlabeled AP-β (Ala)6. However, the shape of Ala 13Cβ peak of AP-β (Ala)2[3-13C]Ala3(Ala)3 looks like a mixture of the peaks of nonlabeled AP-β (Ala)6 and nonlabeled AP-β (Ala)7, implying a mixture of rectangular and staggered packing structures.20 In contrast, a small doublet peak was observed for the Ala 13Cβ carbon of AP-β (Ala)4[3-13C]Ala5Ala. Thus, Ala Cβ peak of the individual G


Article

Macromolecules

Figure 8. A series of partially relaxed 13C CP/MAS NMR spectra of the Ala Cβ peaks of (a) Ala[3-13C]Ala2(Ala)4, (b) (Ala)2[3-13C]Ala3(Ala)3, and (c) (Ala)4[3-13C]Ala5Ala in the dry state at 25 °C as a function of delay time τ for T1 determinations.

main Ala Cβ peak (20.6 ppm) of (a) AP-β Ala[3-13C]Ala2(Ala)4 was 0.24 s, which is slightly larger than the T1 value (0.16 s) of nonlabeled AP-β (Ala)6. There is an additional small peak at 22.0 ppm with T1 = 0.52 s, which indicates the presence of staggered packing. Like spider dragline silk fibers, there are two kinds of packing structures in the sample, (b) AP-β (Ala)2[3-13C]Ala3(Ala)3. The T1 values were 1.38 s (22.7 ppm), 0.17 s (20.6 ppm), and 0.38 s (19.6 ppm) which seem reasonable by assuming T1 values of a mixture of (Ala)6 (rectangular) and (Ala)7 (staggered) as listed in Table 1. Thus, the appearance of the staggered packing structure was confirmed from T1 observation as well as the line shape analysis. The T1 values of two main peaks were 0.28 s (21.2 ppm) and 0.24 s (20.3 ppm), which might indicate the intermediate packing structure between rectangular and staggered arrangements according to the line shape and the T1 values. (Intermediate values of 0.16 s at the 20.6 ppm peak of AP-β (Ala)6 and 0.33 s at 19.6 ppm of AP-β (Ala)7 were found.) There is also an additional small peak at 22.0 ppm with T1 = 0.50 s, which signifies the presence of staggered packing, similar to the case of (a) AP-β Ala[3-13C]Ala2(Ala)4. These results imply that characteristic spectral and dynamical patterns of the staggered packing arrangements arise at the central parts in AP-β (Ala)6 with dominantly rectangular packing arrangement.

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carbons between (Ala)6 and (Ala)7, which correspond to rectangular and staggered packing arrangements, respectively. The Ala Cβ peaks of poly-A regions of spider dragline silks could be reproduced by the superposition of these two peak patterns, and as a result, the fraction of rectangular and staggered packing arrangements could be determined. Another advantage of solid-state NMR is the availability of quantitative dynamical information through selected NMR relaxation measurements such as spin−lattice relaxation.26−43 The motional rates and modes can be obtained. Especially, the hopping motion of Ala Cβ carbons in the poly-A region of spider dragline silks is expected to be influenced strongly by the packing arrangements because the Ala Cβ carbons are located outside the backbone. Thus, in this paper, we tried to prove the presence of the mixture of the rectangular and staggered packing arrangements from the viewpoints of protein dynamics through the determination of T1 values of Ala Cβ peaks of two kinds of spider dragline silks. The difference in the T1 value of the peak of Ala Cβ carbons clearly supported the coexistence of the rectangular and staggered packing arrangements. In particular, the remarkably long T1 values at 22.9 ppm clearly supported the presence of the staggered packing structure in the spider dragline silks. The origin of these long T1 values in the staggered packing structure was clarified from the 13C solid-state NMR relaxation work of S. c. ricini silk fibroin fiber and the MD simulation and interpreted as due to the presence of a geared hopping motion under strong coupling of two methyl groups.43 Earlier, two components with significantly different mobilities were reported for Ala residues of N. clavipes dragline silk fibers from the 13C Ala Cβ peak of the 13C CP/MAS NMR spectrum by Simmon et al.26 They reported 40% immobile component with 13C T1 = 0.18 s, and 60% mobile component with T1 = 2 s from the relaxation analysis of the 13C Ala Cβ peak. From the line shape analysis of the 2H solid-state NMR spectrum of [3,3,3-2H3]Ala-of N. clavipes dragline silk fiber, they also reported two types of poly-A segments: one that is highly oriented (40%) and one that poorly oriented and less densely packed (60%).27 Shi et al.31 also reported two components for [3,3,3-2H3]Ala-N. clavipes dragline silk fiber with 2H spin−lattice relaxation times, where the T1’s were

DISCUSSION

It is important to determine the packing arrangements of AP-β poly-A region in spider dragline silks because it is well-known that the high strength of these fibers come from the poly-A region. In general, such packing arrangements of polymers and proteins have been frequently determined using X-ray diffraction methods, but the spider dragline silks are amorphous-rich samples which give limited information about the packing structures.20 Instead of X-ray diffraction, solid-state NMR is very effective for the structure determination. In our previous paper,20 for the first time we reported the packing arrangements of poly-A regions in spider dragline silks to be a mixture of the rectangular and staggered packing arrangements on the basis of the line shapes of Ala Cβ peaks. In fact, quite different line shapes were observed for Ala Cβ H


Article

Macromolecules measured indirectly using 13C detected 2H−13C CP/MAS NMR in dry and in hydrated states. However, in these solidstate NMR studies, they did not try to assign the Ala Cβ peaks in the 13C CP/MAS NMR spectra of spider silks to the packing structures although the peaks were clearly split into several components. The assignments of the peaks to the packing structures were reported by us for the first time. Thus, two components observed by Simmon et al. and Shi et al. might be due to the differences in the dynamical character of the two kinds of the packing structure of poly-A region of N. clavipes dragline silk fiber. Fossey and Tripathy49 emphasized the presence of interphases rather than the crystalline and amorphous phases to interpret the initial modulus of the spider dragline silk fiber. Paquet-Mercier et al.50 observed the attenuated total reflection infrared spectroscopy of Naphila clavipes dragline silk fiber and showed the presence of two types of β-sheet structures from the data of D2O-inaccessible and -accessible β-sheet deuteration in the amide II region when the fiber samples were immersed in D2O. Sapede et al.51 supported a three-phase model of nanofibrils composed of crystalline and short-range order domains, which are embedded in an amorphous matrix through neutron scattering of Nephila edulis dragline silk fiber. Plaza et al.52 analyzed the mechanical properties of Argiope trifasciata dragline silk fibers in terms of three fractions of the materials, i.e., amorphous, highly oriented nanocrystals, and weakly oriented material with different values of the macroscopic alignment parameters. Recently, Riekel et al.13 studied a hierarchical organization of Argiope. bruennichi whole dragline silk fibers using X-ray nanodiffraction techniques that provided nanoscale step-scanning resolution. The core consists of a composite of several nanometers size crystalline nanodomains with poly-A microstructure, embedded in a polypeptide network with short-range order. Thus, in their works, there is no consideration of two kinds of packing structure of AP-β poly-A regions as possible interpretation of their experimental data for spider dragline silks. The interpretation may change if the additional information about two kinds of packing arrangements is included. In this paper, the overall packing structure of AP-β (Ala)6 was the rectangular arrangement, but the staggered arrangement was observed clearly in the line shape of the Ala Cβ carbon of (Ala)2[3-13C]Ala3(Ala)3. Furthermore, the remarkably longer T1 value of 22.7 ppm peak was also obtained like (Ala)7 and S. c. ricini silk fibroin fibers with the staggered arrangements. These findings are of interest because the length of poly-A region in spider dragline silk fibers is mostly 6 or 7.5,6 Our results reported previously indicate that the packing structure, i.e., the fraction of rectangular and staggered arrangements, changed significantly between the two spider silks, NCF and RSP, as well as the change in the physical states, i.e., dry and hydrated states. Thus, the spiders control the packing structure depending on the species and the hydration of poly-A regions. As a future work, it is important to correlate the different proportions of rectangular, staggered, and random coil of several spider silk fibers with their mechanical properities.

with the sequence from ADF-3 silk protein from the European garden spider A. diadematus. The difference in the T1 value of each peak of Ala Cβ carbons clearly supported the presence of a mixture of the rectangular and staggered packing arrangements. In particular, the remarkably long T1 values at 22.9 ppm supported the presence of the staggered packing structure in the spider dragline silks. The dynamics of both spider silks were studied by T1 in dry and hydrated states. The presence of the staggered packing structure was also confirmed by using [3-13C]Ala-labeled (Ala)6 with the rectangular packing arrangement. Thus, the previous experimental data and their analyses for spider dragline silks reported by other researchers may change if the additional information about two kinds of packing arrangements is included.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b01402. Figure S1: plot of spin−spin relaxation (T1) values as a function of correlation time (τc) values calculated by using eq 1 for Ala Cβ carbon;43 Figure S2: expanded 13C solid-state NMR spectra (10−70 ppm) of [3-13C] N. clavata dragline silk fibers together with the assignments: (a) 13C refocused INEPT, (b) 13C DD/MAS, and (c) 13 C CP/MAS NMR spectra in the hydrated state and (d) 13C CP/MAS spectrum in the dry state, where (β) is β-sheet and (r.c.) is random coil;20 Figure S3: expanded 13 C solid-state NMR spectra (10−70 ppm) of MeOHRSP fiber (black solid line) and MeOH-RSP powder (red broken line) together with the assignments: (a) 13C refocused INEPT, (b) 13C DD/MAS, and (c) 13C CP/ MAS NMR spectra in the hydrated state and (d) 13C CP/MAS spectrum in the dry state, where (β) is β-sheet, (r.c.) is random coil, “t” is trans, and “c” is cis;45 Figure S4: plots of log Mτ vs τ values for the individual peaks in Figures 3 and 4 and Table 2 (here Mτ means the peak intensity at τ s; the T1 values are summarized in Tables 1 and 2); Figure S5: expanded Ala Cβ region in 13C CP/ MAS spectra of recombinant silk protein, RSP with antiparallel β-sheet structure: (a) [3-13C]Ala-RSP spectrum, (b) nonlabeled RSP spectrum, (c) difference spectrum: (a) − (b) (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: 0000-0003-3389-9318 Akira Naito: 0000-0003-2443-6135 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.A. acknowledges support by a JSPS KAKENHI, Grant-in-Aid for Scientific Research (A), Grant JP26248050, and Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT). We also acknowledge Drs. J. Sugahara and T. K. Sato at Spiber Inc. for giving us the recombinant spider silk

CONCLUSION In this work we tried to prove the importance of the mixture of the rectangular and staggered packing arrangements through the determination of T1 values of Ala Cβ peaks in two kinds of spider dragline silks, N. clavate silk and recombinant spider silk, I


Article

Macromolecules

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proteins. T.A. also acknowledges Dr. H. N. Cheng (Southern Regional Research Center, USDA, Agricultural Research Service, New Orleans, LA 70124) for discussions.

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