Toward Understanding the Silk Fiber Structure: 13C Solid-State NMR

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules 13

Towards Understanding Silk Fiber Structure: C Solid State NMR Studies of the Packing Structures of Alanine Oligomers before and after Trifluoroacetic Acid Treatment Tetsuo Asakura, Michi Okonogi, and Akira Naito J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b04565 • Publication Date (Web): 14 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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The Journal of Physical Chemistry

Towards Understanding Silk Fiber Structure: 13C Solid State NMR Studies of the Packing Structures of Alanine Oligomers before and after Trifluoroacetic Acid Treatment

Tetsuo Asakura,* Michi Okonogi and Akira Naito

Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo

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ABSTRACT: Polyalanine (poly-A) sequences with tightly packed antiparallel  sheet (AP-) structures are frequently observed in silk fibers and serve as a key contributor to the exceptionally high fiber tensile strength. In general, the poly-A sequence embedded in the amorphous glycine-rich regions has different lengths depending on the fiber type from spiders or wild silkworms. In this paper, the packing structures of AP- alanine oligomers with different lengths were studied using 13C solid-state NMR as a model of the poly-A sequences. These included alanine oligomers with and without the protection groups (i.e., 9-fluorenylmethoxycarbonyl and polyethylene glycol groups at the N- and C-terminals, respectively). The fractions of the packing structures as well as the conformations were determined by deconvolution analyses of the methyl NMR peaks. Trifluoroacetic acid was used to promote the staggered packing structures, and the line　 shapes changed significantly for oligomers without the protected groups but only slightly for oligomers with the protected groups. Through NMR analysis of the 3-13C singly labeled alanine heptamer and refined crystal structure of the staggered packing units, a possible mechanism of the staggered packing formation is proposed for the AP alanine heptamer.


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INTRODUCTION Polyalanine (poly-A) sequences are frequently observed in spider dragline silks1-6 and wild silkworm silks.7-14 The origin of the exceptionally high tensile strength of these silk fibers has been attributed to the crystalline antiparallel  sheet (AP-) structures formed by the repeating poly-A sequences embedded in the amorphous glycine (Gly)-rich regions.1-5, 15,16 In general, the length of poly-A sequences is different depending on the different silk fibers from several kinds of spiders1-3 and wild silkworms.7,12,13 An example can be shown for the poly-A sequences present in major (Ma) ampullate and minor (Mi) ampullate silks from the spider Nephila clavipes.

Both

Ma and Mi silks are composed of two proteins, MaSp1 and MaSp2.2,15,17 One distinct difference between these two silks is the number n in the poly-A sequence ((Ala)n), up to 7 in MaSp1 and 10 in MaSp2 for Ma, while Mi has shorter poly-A sequences with n=3-5 in MaSp1/MaSP2.2,17-19 In contrast, the number n for the repeat poly-A sequences embedded in the amorphous Gly-rich regions is mainly 12 or 13 for silks from wild silkworm, such as Samia cynthia ricini (S.c.ricini),9,10,12 Antheraea pernyi,7Antheraea yamamai 13and Antheraea mylitta.8 Thus, it is important to further clarify the packing structures of alanine oligomers with AP- structures depending on the poly-A length. The packing structures of AP- (Ala)3 and AP- (Ala)4 were reported using singlecrystal X-ray diffraction analysis because single crystals of these peptides with enough size for X-ray diffraction analysis could be obtained.20-23

For example, we had

reported the atomic co-ordinates of the single crystal AP- (Ala)4 sample as shown in the Supporting Information, Figure S-1.22,23

The AP- (Ala)4 molecules were aligned

in the head-to-tail fashion with methyl groups arranged alternately above and below the



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plane of the AP- sheets. Water molecules were bridged between adjacent N and C termini. The strands were packed into a rectangular lattice and formed hydrogen bonds side-by-side as well as end-to-end. The end-to-end interactions occurred through the bridging water molecules. Thus, the rectangular packing arrangement of the (Ala)4 molecules was stabilized by the intermolecular hydrogen bond formation among water molecules and the charge groups at the N- and C-terminal ends. However, for longer AP- (Ala)n samples with n ≥5, we could not obtain single crystals with sufficient size for X-ray diffraction. Therefore, we used mainly 13C solid-state NMR to determine the packing structures of a series of alanine oligomers with different alanine lengths, n ≥5.22-28 As summarized in the Supporting Information, Figure S-2, we found out from the significant line shape changes in the Ala C peaks that the packing structures changed depending on the alanine lengths.22,25,28 In particular, the 13C chemical shifts of Ala C peaks of the inner three Ala residues in AP- (Ala)5 and also that of a single (apparent) methyl peak of the AP- (Ala)6 were in good agreement with the averaged 13C chemical shifts of the internal Ala2 and Ala3 residues in AP- (Ala)4. Therefore, the packing structures of AP- (Ala)5 and AP- (Ala)6 were speculated to consist mainly of rectangular arrangements. In comparison, the 13C NMR spectral patterns of AP- (Ala)7 and longer alanine oligomers had two peaks with the chemical shifts, 22.7 ppm and 19.6 ppm, which were quite different from that of AP- (Ala)6. This indicated a change in the poly-A chain packing pattern from rectangular arrangement to staggered arrangement because Arnott et al.29 reported that AP- poly-A samples with high molecular weight took on the staggered packing arrangement.

Thus, the factors that 4

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stabilized the packing structures changed depending on the alanine lengths. The presence of water molecules and/or the charged groups at the N- and C-terminal ends of poly-A molecules in alanine oligomers was undoubtedly important factors. Actually, by heating AP- (Ala)6 at 200 °C for 4 h, the Ala C peak patterns changed from rectangular packing to staggered packing due to the removal of water.25 Therefore, it is useful to examine the packing structure of AP- alanine oligomers without the presence of water molecules. Instead of water, trifluoroacetic acid (TFA) seems to be suitable for the examination of the packing arrangement because longer alanine oligomers, (Ala)n (n ≥5) are soluble in TFA.30,31 In addition, the packing structure needs to be further studied for the alanine oligomers with the protected groups at both ends.32,33 In this paper, the packing arrangements of Ala oligomers, (Ala)n (n=3-7,12) are examined before and after TFA treatment using 13C solid-state NMR. In addition, in order to remove the effect of charges at both N- and C-terminal, the samples with protection groups [the N-terminus being blocked by the 9-fluorenylmethoxycarbonyl (Fmoc) group and the C-terminus blocked by polyethylene glycol (PEG) group] are also used for the 13C solid-state NMR analysis.33

These modified alanine oligomers are

especially useful for (Ala)n (n=7,8 and 12), as a way to examine the staggered packing structure in detail.

Since we have already reported new atomic co-ordinates of the

staggered packing arrangement by modification of Arnott structural model for AP- poly-A chains,34-36 the elucidation of the staggered packing structure through 13C solidstate NMR analyses can be done on the basis of these atomic co-ordinates. As an example, free and protected (Ala)7 peptides with only 13C methyl labeled at the central Ala residue are studied by 13C solid-state NMR (including 13C solid-state NMR relaxation experiments) in order to examine in detail the staggered packing structure. 5 ACS Paragon Plus Environment


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MATERIALS AND METHODS Alanine oligomers. The Ala oligomers, (Ala)n (n=3-7,12) and (Ala)3[313C]Ala(Ala) prepared 3

previously22,25 were dissolved in TFA and then dried (TFA

treatment). Fmoc(Ala)n (n=7, 8 and 12)PEG and Fmoc(Ala)3[3-13C]Ala(Ala)3PEG were synthesized by a PioneerTM Peptide Synthesizer using Fmoc solid phase method as reported previously.33 These samples were dissolved in 55%(w/w) lithium thiocyanate (LiSCN) and then dialyzed against ion-exchanged water for three days. The precipitated peptides were finally collected by lyophilization for 13C solid-state NMR measurements of the peptides before TFA treatment together with the peptides after TFA treatment. The purity of all peptides was more than 95%, as verified by high-performance liquid chromatography, 13C solution NMR, and IR. [3-13C]Ala (99.9% 13C enrichment) was purchased from Cambridge Isotopes Laboratories, Andover, MA, USA.

Solid-state 13C CP/MAS NMR Measurements.

13C

CP/MAS NMR spectra of all

peptides were recorded on Chemagnetics Infinity 400 and Bruker Avance 400 NMR spectrometers. In both cases of NMR observation, 7 kHz of MAS spinning speed, 3 ms of CP contact time, and 5 sec of recycle delay were applied.

The number of scans was

15,360 for (Ala)n (n=3-7,12) after TFA treatment, 3,072 for Fmoc(Ala)nPEG (n=7 and 8), and 7,168 for Fmoc(Ala)12PEG before and after TFA treatment, and 150 scans for the 13C labeled peptides. The 13C chemical shifts were referenced to TMS using adamantane as a secondary standard (13CH peak at 28.8 ppm). The 13C solid-state spinlattice relaxation time (13C T1) was determined for (Ala)3[3-13C]Ala(Ala)3 and Fmoc(Ala)3[3-13C]Ala(Ala)3PEG before and after TFA treatment by the method of Torchia37 using Chemagnetics Infinity 400 spectrometer. The delay times were varied at 6 ACS Paragon Plus Environment

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the range of 0.01 to 3 sec for the T1 measurements of the Ala Cβ peaks.27 The fractions of the conformations and packing structures of Ala residues of all peptides were determined from the deconvolution of the Ala C peaks.28,38 The error was about ±2% for each fraction in the deconvolution analysis after several deconvolution trials.

RESULTS 13C

CP/MAS NMR spectra of (Ala)n (n=3-7 and 12) after TFA treatment.

Figure 1 shows 13C CP/MAS NMR spectra of (Ala)n (n=3-7 and 12) after TFA treatment. In the (Ala)3 spectrum, three peaks were observed for the Ala C carbons, two asymmetric peaks for Ala C carbons and several peaks for Ala C=O carbon. The chemical shifts of these peaks were different from those of random coil and also dissimilar to AP- sheet structure unless parallel (P)- sheet structure was considered.2224

Thus, (Ala)3 after TFA treatment seems to take on a special structure. The Ala C and

Ala C peak patterns of the spectrum of (Ala)4 after TFA treatment were similar to those of P- (Ala)4 but different from AP- (Ala)4 although the Ala C=O peak splits into several peaks.39 This indicates that (Ala)4 after TFA treatment approximately takes on the P- sheet structure although it deviates slightly from a typical P- (Ala)4 structure. However, we did not try to delve more into the structures of (Ala)3,4 because we preferred to concentrate mainly on the staggered packing structures of longer alanine oligomers. In contrast to these two peptides, (Ala)5 after TFA treatment seemed to take on mainly the AP- sheet structure. Thus, the chemical shift of the main Ala C peak was 19.8 ppm, that of Ala C single peak was 49.5 ppm, and that of slightly broad Ala C=O peak was around 172 ppm. Moreover, the 13C CP/MAS NMR spectra of (Ala)6,7,8 7 ACS Paragon Plus Environment


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after TFA treatment were similar to one another and showed the typical AP- sheet structures. The Ala C peaks of (Ala)n (n=5,6,7 and 12) after TFA treatment are expanded in Figure 2 (left) together with the peak deconvolution analysis for elucidation of the packing structure. The fractions determined by the deconvolution analyses are summarized in Table 1, and the pie charts of the fractions are also shown in Figure 2 (right). The Ala C peaks of (Ala)n (n=5,6 and 7) were deconvoluted to four peaks: ① 22.7±0.2 ppm, ②21.2±0.2 ppm, ③19.8±0.1 ppm, and ④16.9±0.4 ppm with different half-height widths.28,38 The peak pattern with a combination of two peaks at 22.7 ppm and 19.8 ppm was assigned to the staggered packing arrangement of AP- sheet structure as reported in our previous papers.22,25,28 One peak at 21.2 ppm obtained from the peak deconvolution analysis was assigned to rectangular packing arrangement of AP- sheet structure, as reported previously.22 However, all of the other -sheet structures except for the contributions to the staggered packing structure could possibly be associated with 21.2 ppm. The highest field broad peak at 16.9 ppm in the Ala C peaks of (Ala)n (n=5,6 and 7) was assigned to random coil. Furthermore, for (Ala)12, one additional highest field small peak at 15.3±0.7 ppm was observed although the fraction was small, suggesting the appearance of a small amounts of -helix conformation.30,31 A significant difference was observed in the Ala C peaks of (Ala)5 and (Ala)6 after TFA treatment compared with the previous Ala C peaks observed for the precipitated peptides obtained after dialyzing the concentrated LiSCN solutions against water (Figure S2). Thus, the main peaks were observed at 19.8 ppm together with the small 8 ACS Paragon Plus Environment

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peaks at 22.7 ppm for these peptides after TFA treatment, indicating the formation of staggered packing structure. Only small fractions of the 21.2 ppm peak were obtained. Thus, it is clear that the appearance of the rectangular packing structures observed for AP- (Ala)5 and AP- (Ala)6 as described previously22, 25was due to the hydrogen bond formations between terminal alanine residues via water molecules. With increasing n in (Ala)n, the fraction of 19.8 ppm decreased gradually.

Fractions of the different structures of Fmoc(Ala)nPEG (n=7,8 and 12) before and after TFA treatment determined from the deconvolution analysis of the Ala C peaks. The expanded Ala C peaks of (Ala)n and Fmoc(Ala)nPEG (n=7,8 and 12) were shown in Figures 3-5 (left), respectively, together with those of Fmoc(Ala)nPEG after TFA treatments. The fractions determined by the deconvolutions analyses were summarized in Table 1 and the pie charts of the fractions were also shown in Figures 35 (right). The effect of the protecting groups at both ends as well as the effect of TFA treatment on the staggered packing structures were studied. As shown in Supporting Information, Figure S-3, the 13C CP/MAS NMR spectra of the protected peptides indicated the presence of the terminal-blocked groups due to the observation of small broad peaks at 120-130 ppm, 140-145 ppm, and 155 ppm from the Fmoc group, and a small peak at 70ppm from the PEG group.32

The 13C chemical shifts of Ala C, C

and C=O peaks were 19.8 ppm (main peak), 49.0 ppm and 172 ppm, respectively, suggesting that the conformation was still the AP -sheet structure although the protecting groups of both Fmoc and PEG were attached at both ends.40 As a result of the presence of the Fmoc and PEG groups at the both ends, the fractions of 21.2 ppm and 9 ACS Paragon Plus Environment


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16.9 ppm peaks increased very little for (Ala)7 and (Ala)8, indicating a small disturbance of the staggered packing structure due to the protected groups. With TFA treatment of Fmoc(Ala)7PEG, the fraction of the 19.8 ppm peak increased, while that of 21.2 ppm decreased significantly. These tendencies were also observed for Fmoc(Ala)8PEG as shown in Figure 4. The differences in the fractions between (Ala)12 and Fmoc(Ala)12PEG were not so large as the former two cases except for the significant increase of the fraction of -helix peak at 15.3 ppm for Fmoc(Ala)12PEG (Figure 5). Owing to TFA treatment, the fraction of 19.8 ppm peak increased and those of other peaks decreased slightly.

Comparison of the fractions of the structures determined from the 13C CP/MAS NMR spectra of (Ala)3[3-13C]Ala(Ala)3, and Fmoc(Ala)3[313C]Ala(Ala)

3PEG

before and after TFA treatments

The presence of the protected groups or TFA treatment was also expected to change the structure of Ala residues depending on the position in alanine oligomers. In order to obtain such an information, the methyl carbon of the central Ala residue in (Ala)7 was selectively 13C labeled. Figure 6 (left) showed the Ala 13C peaks of (a) (Ala)3[313C]Ala(Ala) , 3

(b) Fmoc(Ala)3[3-13C]Ala(Ala)3PEG and (c) Fmoc(Ala)3[3-

13C]Ala(Ala) PEG 3

after TFA treatment together with the pie charts of the fractions

(Figure 6, right). In comparison with three Ala C peaks in Figure 3, the random coil peak at 16.9 ppm was essentially absent for all three Ala C peaks. This came from two causes: first, the central Ala residue in (Ala)7 contributed to tightly staggered packing structure, and secondly, random coil occurred near the end Ala residues in alanine oligomers. Other characteristics included the significantly sharp peaks, especially the 10 ACS Paragon Plus Environment

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19.8 ppm peak, suggesting that the chemical shift distribution observed at the 19.8 ppm peak in Figure 3 became remarkably small as evidenced by the methyl group of the central Ala residue in (Ala)7. In addition, a significant increase in the fraction of the 21.2 ppm was observed when the protected groups were attached to the N- and Ctermini of (Ala)7 (Figures 6 (a) and 6 (b)). In contrast, with TFA treatment, a significant decrease in the fraction of the 21.2 ppm peak and a significant increase in that of the 19.8 ppm peak were observed (Figure 6 (b) and (c)). A more detailed discussion on the staggered packing structure of (Ala)7 is possible by using the fractions determined from both Figures 3 and 6, as will be done in the Discussion section.

13C

CP/MAS NMR spin-lattice relaxation times of (Ala)3[3-13C]Ala(Ala)3, and

Fmoc(Ala)3[3-13C]Ala(Ala)3PEG before and after TFA treatments In order to confirm the appearance of the staggered packing structures for (Ala)7 when the protected groups, Fmoc and PEG, were attached or after TFA treatment, 13C solid-state NMR T1 values were obtained for the 13C singly labeled Ala C carbons of (Ala)3[3-13C]Ala(Ala)3 and Fmoc(Ala)3[3-13C]Ala(Ala)3PEG before and after TFA treatments.26,27,36 Figure 7 shows a series of partially relaxed 13C solid state NMR spectra of the Ala 13C peaks during T1 relaxation as a function of delay time τ. The T1 values (in msec) of the peaks at 22.7 ppm, 21.2 ppm and 19.8 ppm were 1205±55, 588±26 and 669±22 for (Ala)3[3-13C]Ala(Ala)3, 1182±23, 538±21 and 630±19 for Fmoc(Ala)3[3-13C]Ala(Ala)3PEG before TFA treatment, and 1142±22, 414±14 and 647±33 for Fmoc(Ala)3[3-13C]Ala(Ala)3PEG after TFA treatment, respectively. Thus, the longest T1 value was obtained for the peak at 22.7 ppm, at almost twice the other two peaks. The same tendencies were observed for three kinds of [3-13C]Ala carbons. 11 ACS Paragon Plus Environment


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Such a long T1 value for 22.7 ppm peak was already observed and interpreted by us in terms of unusually fast “geared hopping” motion in methyl groups.27,36 The unusual motion of the methyl carbon of the central Ala residue in (Ala)7 was not affected by the presence of the protected groups or the TFA treatment.

DISCUSSION The silk structures of spiders and wild silkworms in the solid state have been previously investigated by many spectroscopic techniques including IR41 and Raman4247,

X-ray diffraction,13,48-57 and solid-state NMR.9,18,24-28,30,31,34-36,38-40,56,58-77 These have

provided many insights into the molecular structure of these silk samples. IR and Raman have been used frequently to calculate the fractions of the conformations of silk structure with empirical methods, but it may be difficult to determine the packing of the -sheet structure with IR and Raman. If enough size of the single crystal of silk sample can be obtained, single-crystal X-ray method is undoubtedly a most powerful technique to determine the atomic co-ordinates of silk samples, including the packing structure. Whereas Raman can permit the size and orientation of -sheet crystallites in the samples to be determined, in general it seems difficult to use Raman to determine the packing structures of amorphous-rich polymer samples such as silk fibers. At present, only 13C solid state NMR can give the definitive information on the packing structures of poly-A domains through the analysis of the Ala C peaks. In our previous papers, 34,35,78,79 we determined the packing structures of AP- Bombyx mori and S.c.ricini silk fibroin fibers using solid-state NMR by focusing on the Ala C peaks because these methyl groups are located outside the backbone chains and are sensitive to the packing structure. In this paper, we took advantage of solid-state 12 ACS Paragon Plus Environment

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NMR spectroscopy coupled with the synthesis of the appropriate alanine oligomers to determine the packing structure and showed that AP- poly-A sequences embedded in the Gly-rich regions of silk fibers mostly took on the staggered packing structure. Moreover, the possible mechanism for the formation of staggered packing of alanine oligomers could be derived from the refined crystal structure of staggered packing units.34 As shown in Figure 8,34, 36 four Ala residues (①, ②, ③ and ④) in the upper AP- sheet interacted with four Ala residues (①*, ②*, ③* and ④*) in the lower AP sheet to form staggered unit. In particular, C-Ala③ strongly interacted with C-Ala ③* as evidenced by the long 13C T1 value of C carbon nuclei (also shown in Figure 7) and by theoretical MD simulations.36 In case of (Ala)7, a block with 4 Ala residues in the upper AP- sheet layer interacted with another block with 4 Ala residues in the lower AP- sheet, both stacked in the staggered arrangement as shown in Figure 9 (right). One staggered unit was formed from 8 Ala residues. In case of (Ala)7 after TFA treatment, 3 staggered units were formed in the stacked 2 AP- sheets in the head-to-tail arrangement (Figure 9A1) and one residue in a shifted arrangement (Figure 9B1). In one staggered unit, C carbon of An* in the upper AP- sheet strongly interacted with another C carbon of An’* in the lower AP- sheet and gave a 13C chemical value of 22.7 ppm, observed as a lower field peak in the staggered unit (SGL). This inter -sheet C-C interaction was revealed to be very strong because the 13C T1 value of the C carbon showed the longest value among C carbons in the staggered unit42 because the C-C distance was very short.34 In other words, H(C③) and H(C③*) strongly interacted between two methyl groups to show a geared type motion. Other three C carbons of A residues in the staggered unit gave the 13C chemical shift value of 19.8 13 ACS Paragon Plus Environment


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ppm observed as the higher field peak in the staggered unit (SGH). These C carbons also showed inter-sheet interactions as revealed by our previous work.36 In contrast, A1 and A7 residues in the head-to-tail stacking arrangement formed a -sheet structure but did not exhibit staggered packing. A1 and A7 residues in the one-residue-shifted stacking arrangement formed the -sheet but did not exhibit inter-sheet interactions. Therefore, the 13C chemical shift value was 21.2 ppm, characteristic of -sheet () structure, which was similar to the rectangular type of AP- sheet packing structure. In addition to the stacked -sheets which formed equal amounts of staggered units, it might be possible to form AP- sheet without (or with partial) inter-sheet interactions, which did not contribute towards staggered unit formation. In this case, the ratio of 13C NMR peak intensities for C carbon of SGL to SGH was 1:3 for the staggered AP sheet unit, as shown in Figure 9(middle), which agreed well with the experimentally obtained ratio of 1:2 (Table 1) for Fmoc(Ala)7PEG after TFA treatment. For the case of Fmoc(Ala)3[3-13C]Ala(Ala)3PEG after TFA treatment, the ratio of SGL to SGH was 1:3 for head-to-tail packing arrangement (Figure 9A1, middle) and 1:1 for one-residue-shifted packing arrangement (Figure 9B1, middle). When the two packing arrangements were equally formed, the ratio of SGL to SGH was 3:5. This again showed a good agreement with the experimentally obtained value of 3:4 (Table 1). In the case of Fmoc(Ala)7PEG before TFA treatment, experimentally obtained ratio of SGL (22.7 ppm):SGH (19.8 ppm) was 1:1.6 (Table 1), which was smaller than the predicted values of 1:3 for the staggered AP- sheet unit. In this protected alanine oligomer, the edge part of staggered unit might be disturbed by the protected groups to form inter-sheet interaction. Consequently, the edge part of staggered unit might not 14 ACS Paragon Plus Environment

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form a basic staggered unit, but formed pseudo-staggered unit in which the SGL peak kept the same chemical shift value, while the SGH peak might shift to the  structure because of the weak inter-sheet interactions as shown in Figure 9A2 (left) and B2 (left). In this case, the ratio of SGL:SGH became 1:2, which agreed better with the experimentally obtained value of 1:1.6. For the case of Fmoc(Ala)3[3-13C]Ala(Ala)3PEG before TFA treatment, the labeled position (colored green) was located in the central part of the three staggered units and the ratio of SGL:SGH was 3:5, which agreed better with experimentally obtained value of 1:1. As shown in Figure 6 (middle), the fraction of  peak before TFA treatment was larger than that after TFA treatment. The  peak for the labeled poly-A oligomers could be considered to come from the isolated or the surface part of AP- sheets (Figure 9C). These sheets contained no (or partial) inter-sheet interactions, and hence the labelled position might show  peaks in the center part or in the random coil (r. c.) at the terminal ends. It appeared that the number of AP- sheet to form staggered packing structure was smaller than that after TFA treatment. It was also compatible with the fact that the labelled Ala C did not show random coil peaks because the labeled Ala C was located in the center part of -sheet. Thus, Fmoc(Ala)7PEG after TFA treatment might cause the formation of many staggered units rather than pseudo-staggered unit. In addition, the stacking number of AP- sheet should be larger than that before TFA treatment because the ratio of  peak was very small. This indicated that Fmoc(Ala)7PEG treated by TFA might form stronger inter-sheet interactions which might engender higher toughness in the fibril state. In contrast, for Fmoc(Ala)7PEG before TFA treatment the edge part of the 15 ACS Paragon Plus Environment


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staggered unit might form pseudo-staggered unit to give weak inter-sheet interactions, which might result in reduced toughness in the fibril state.

CONCLUSIONS In this work, the fractions of the conformations and the packing structures of a series of alanine oligomers were determined from deconvolution analyses of the line shapes of the Ala C carbons in the 13C solid-state NMR spectra. The fractions of the peaks at 19.8 and 22.7 ppm were preponderant in all samples studied here, which meant that the predominant packing structure was the staggered conformation. The dominant packing structures of AP- (Ala)5 and AP- (Ala)6 were rectangular when these peptide samples were prepared as the precipitated powder dialyzed from the concentrated LiSCN aqueous solution against water,22 but the structures changed to the staggered form after TFA treatment. Thus, for (Ala)5 and (Ala)6, the staggered packing structure was formed during the process of removal of TFA by drying via the random coil conformation in the TFA solution. For longer AP- (Ala)n samples with n ≥7, the staggered packing structures were always dominant, and the presence of the protected groups at both ends had only a minor effect on the fraction of each peak of the Ala C groups. For (Ala)12 and Fmoc(Ala)12PEG before and after TFA treatment, an additional peak at 15.3 ppm (most upfield) was observed, suggesting the appearance of an -helix conformation although the fraction of this conformation is small. Thus, some disturbance seems to occur at the end groups in the process of forming the staggered packing structure because of the presence of protected groups at the ends or after TFA treatment. By using 13C

solid-state NMR analyses {including the solid-state NMR relaxation studies of

(Ala)3[3-13C]Ala(Ala)3, Fmoc(Ala)3[3-13C]Ala(Ala)3PEG and Fmoc(Ala)3[316 ACS Paragon Plus Environment

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


13C]Ala(Ala) PEG 3

after TFA treatment}, the possible mechanism for staggered packing

formation of AP- alanine heptamer is proposed, and it is compatible with the refined crystal structure of staggered packing units.29,34

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI: -----Figure S1. Rectangular packing structure of A-(Ala)4 determined by single crystal X-ray diffraction analysis.22 Figure S2. Expanded 13C CP/MAS NMR spectra of the Ala C region of (Ala)n (n=3-7 and 12).22 Figure S3. 13C CP/MAS NMR spectra of (a) Fmoc(Ala)7PEG, (b) Fmoc(Ala)8PEG, and (c) Fmoc(Ala)12PEG.

AUTHOR INFORMATION Corresponding Author *(T.A.) E-mail: [email protected] ORCID Tetsuo Asakura: 0000-0003-4472-6105 Akira Naito: 0000-0003-2443-6135 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS T.A. acknowledges support via a JSPS KAKENHI Grant-in-Aid for Scientific Research (C), Grant Number JP19K05609. T.A. also acknowledge Dr. H. N. Cheng (Southern 17 ACS Paragon Plus Environment


Page 18 of 36

Regional Research Center, USDA, Agricultural Research Service, New Orleans, LA 70124, U.S.A.) for discussion.

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Figure 1 13C CP/MAS NMR spectra of (Ala) (n=3-7 and 12) after TFA treatment. n



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Figure 2 (Left) Expanded 13C CP/MAS NMR spectra of the Ala C regions of (a) (Ala)5 , (b) (Ala)6 , (c) (Ala)7 and (d) (Ala)12 after TFA treatment. The deconvoluted spectra are shown as broken lines. (Right) Pie charts of the fractions determined by the deconvolutions. The color of the fraction is ①red (22.7±0.2 ppm), ②green (21.2±0.2 ppm), ③pink (19.8±0.1 ppm), ④light blue (16.9±0.4 ppm) and ⑤blue (15.3±0.7 ppm).


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Figure 3 (Left) Expanded 13C CP/MAS NMR spectra of the Ala C regions of (a)(Ala)7, (b) Fmoc(Ala)7PEG and (c) Fmoc(Ala)7PEG after TFA treatment. The deconvoluted spectra are shown as broken lines. (Right) Pie charts of the fractions determined by the deconvolutions. The color of the fraction is ①red (22.7±0.2 ppm), ②green (21.2±0.2 ppm), ③pink (19.8±0.1 ppm) and ④light blue (16.9±0.4 ppm).



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Figure 4 (Left) Expanded 13C CP/MAS NMR spectra of the Ala C regions of (a)(Ala)8, (b) Fmoc(Ala)8PEG and (c) Fmoc(Ala)8PEG after TFA treatment. (Right) Pie charts of the fractions determined by the deconvolutions. The color of the fraction is ①red (22.7±0.2 ppm), ②green (21.2±0.2 ppm), ③pink (19.8±0.1 ppm) and ④light blue (16.9±0.4 ppm).


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Figure 5 (Left) Expanded 13C CP/MAS NMR spectra of the Ala C regions of (a)(Ala)12, (b) Fmoc(Ala)12PEG and (c) Fmoc(Ala)12PEG after TFA treatment. The deconvoluted spectra are shown as broken lines. (Right) Pie charts of the fractions determined by the deconvolutions. The color of the fraction is ①red (22.7±0.2 ppm), ②green (21.2±0.2 ppm), ③pink (19.8±0.1 ppm), ④light blue (16.9±0.4 ppm) and ⑤blue (15.3±0.7 ppm).



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Figure 6 (Left) Expanded 13C CP/MAS NMR spectra of the Ala C regions of (a) (Ala)3[313C]Ala(Ala) , (b) Fmoc(Ala) [3-13C]Ala(Ala) PEG and (c) Fmoc(Ala) [33 3 3 3 13C]Ala(Ala) PEG after TFA treatment. (Right) Pie charts of the fractions determined 3 by the deconvolutions. The color of the fraction is ①red (22.7±0.1 ppm), ②green (21.2±0.1 ppm) and ③pink (19.8±0.1 ppm).


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Figure 7 A series of partially relaxed 13C solid state NMR spectra of Ala C peaks of (a) (Ala)3[313C]Ala(Ala) , (b) Fmoc(Ala) [3-13C]Ala(Ala) PEG and (c) Fmoc(Ala) [33 3 3 3 13C]Ala(Ala) PEG after TFA treatment as a function of delay time τ. The T values (in 3 1 msec) of the peaks at 22.7 ppm (III), 21.2 ppm (II) and 19.8 ppm (I) were 1205±55, 588±26 and 669±22 for (Ala)3[3-13C]Ala(Ala)3, 1182±23, 538±21 and 630±19 for Fmoc(Ala)3[313C]Ala(Ala) PEG before TFA treatment, and 1142±22, 414±14 and 647±33 for 3 Fmoc(Ala)3[3-13C]Ala(Ala)3PEG after TFA treatment, respectively.


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Figure 8 The model structure of poly-A sequence with AP- structure in S.c.ricini silk fibroin in a staggered packing arrangement proposed by us.34, 36 (a) The fiber axis (c axis) and (b) hydrogen bonding axis. The Ala C carbons with different environments were noted as from ① to ④, and also from ①* to ④*. The inter-molecular direct hydrogen bonding pairs of NH…O=C bonds were noted as from I to IV, and also from I’ to IV’. The squares surrounded by solid lines are the staggered unit cells.



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Figure 9 Formation of staggered packing structures of Fmoc(Ala)7PEG after (A1 and B1) and before (A2 and B2) TFA treatment. (A1and A2) right: Two AP- sheets (upper(U) and lower(L)) stacked with head-to-tail arrangement. (B1 and B2) right: Two AP- sheet (U and L) stacked with one-residue-shifted arrangement. (A1, A2, B1 and B2) left: Components of staggered (SG) and pseudo-staggered (PSG) units. Staggered units consisted of four Ala residues in upper AP- sheet and four Ala residues in lower AP- sheet. SGL, SGH,  and random coil (r. c.) indicated that 13C NMR peaks of the Ala residues appeared at 22.7, 19.8, 21.2 and 16.9 ppm, respectively. (A1, A2, B1 and B2) middle: 13C NMR peak positions and intensities for Fmoc(Ala)7PEG and Fmoc(Ala)3[313C]Ala(Ala) PEG. 3

(C) right: isolated AP- sheet without stacking. (C) left: Components of isolated AP- sheet. (C) middle: 13C NMR peak positions and intensities for Fmoc(Ala)7PEG and Fmoc(Ala)3[3-13C]Ala(Ala)3PEG.


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Table 1 Fractions of several conformations and packing structures determined by peak deconvolution by fixing the 13C chemical shifts and changing the half-height-widths in the Ala C peaks of several alanine oligomers. Sample

①

(Ala)5 after TFA treatment (Ala)6 after TFA treatment (Ala)7 after TFA treatment (Ala)12 after TFA treatment (Ala)7 Fmoc(Ala)7PEG Fmoc(Ala)7PEG after TFA treatment (Ala)8 Fmoc(Ala)8PEG Fmoc(Ala)8PEG after TFA treatment (Ala)12 Fmoc(Ala)12PEG Fmoc(Ala)12PEG after TFA treatment (Ala)3[3-13C]Ala(Ala)3 Fmoc(Ala)3[3-13C]Ala(Ala)3PEG Fmoc(Ala)3[3-13C]Ala(Ala)3PEG

②

③

④

8.8 17.7 23.2 20.9

5.6 7.7 10.5 11.6

63.0 61.9 55.1 48.7

22.6 12.7 11.2 13.0

30.7 27.6 26.7

15.9 17.2 7.4

44.5 44.1 53.4

8.9 11.1 12.5

29.7 26.1 23.2

12.7 14.9 5.8

46.1 46.4 56.8

11.4 12.6 14.2

29.4 25.1 20.3

12.7 13.0 11.1

43.1 39.5 50.6

13.1 15.8 12.6

40.9 37.1 37.3

20.0 27.9 14.3

39.1 35.0 48.4

⑤

5.8

1.6 6.6 5.4

after TFA treatment

The chemical shifts used for the peak deconvolution:①:staggered packing (22.7ppm) ② : -sheet without packing and/or rectangular packing (21.2 ppm) ③ : staggered packing (19.8ppm) ④: random coil (16.9 or 17.3 ppm) ⑤: -helix (15.3 ppm). The error was ±2% for each fraction in the deconvolution analysis after several deconvolution trials.



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TOC Graphic Towards Understanding Silk Fiber Structure: 13C Solid State NMR Studies of the Packing Structures of Alanine Oligomers before and after Trifluoroacetic Acid Treatment

Tetsuo Asakura,* Michi Okonogi and Akira Naito

Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo


Toward Understanding the Silk Fiber Structure: 13C Solid-State NMR

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