Mixture of Rectangular and Staggered Packing Arrangements of

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

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 Tetsuo Asakura,* Yugo Tasei, Akihiro Aoki, and Akio Nishimura Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan S Supporting Information *

ABSTRACT: For the first time, we determined the relative percentages of “rectangular” and “staggered” packing arrangements in the crystalline polyalanine regions with antiparallel β-sheet structure within spider dragline silk fiber from Nephila clavata (NCF) and recombinant silk protein (RSP). The methods used included X-ray diffraction and 13C NMR coupled with selective 13C isotope labeling of the Ala Cβ carbons. From deconvolution analyses of the Ala Cβ peaks in the 13C CP/MAS NMR spectra, the relative percentages of the rectangular arrangements in [3-13C]AlaNCF were determined to be 49 ± 8% and 40 ± 7% in the dry and hydrated states, respectively, and in [3-13C]Ala-RSP 62 ± 11% and 81 ± 5% in the dry and hydrated states, respectively. Thus, the packing structure changed significantly between the two spider silks and also between the two physical states. The use of NMR was critical in this analysis; from X-ray diffraction patterns alone it would have been difficult to obtain these quantitative data.

■

Type I β-turn,22,44 and partially β-sheet23,33,38,39,41 together with their fractions. In this work we have concentrated on the packing structure of poly-A sequences with β-sheet in the spider dragline silks because there have been no detailed reports on this topic thus far. For the 13C Ala Cβ peak in the 13C CP/MAS NMR spectrum, Simmons et al.19 reported two components with significantly different mobilities for Ala residues in N. clavipes dragline silk fiber, viz., 40% immobile component with 13C spin−lattice relaxation times T1 = 0.18 s and 60% mobile component with T1 = 2 s. They also reported two types of polyA sequencesone highly oriented and another poorly oriented and less densely packedfrom the line shape analysis of the 2H solid-state NMR spectrum of [3,3,3-2H3]Ala-N. clavipes dragline silk fiber.20 In contrast, the oriented [1-13C]Ala spider dragline silk fibers were used to analyze the backbone orientation of Ala residues on the basis of large chemical shift anisotropies (CSA) of the carbonyl groups by van Beek et al.23 and Bonev et al.41 Poly-A regions in the oriented dragline silk had a strongly oriented β-sheet structure with the chains predominantly parallel to the fiber together with random coil structures which gave powder X-ray patterns. Although other solid-state NMR techniques, e.g., 2D spin-diffusion NMR (2D CSA tensor correlation through spin diffusion),21,40,42 13C−13C dipolar assisted rotational resonance (DARR),28−31 rotational echo double resonance (REDOR),43,44 and 2H MAS NMR,45 were

INTRODUCTION There are a variety of spiders and silkworms that produce silk fibers with high strength and high toughness. Recently, researchers in many fields, such as biology, biochemistry, biophysics, analytical chemistry, polymer technology, textile technology, and biomaterials, have been interested in the spider dragline silk because of the exceptionally high tensile strength and toughness of the spiders’ major ampullate silk fibers.1−5 Its excellent physical properties appear to result from a combination of the design of its structural proteins (Spidroins) and the controlled processing.2 The dragline silk is secreted by the major (Ma) ampullate gland and is composed mainly of two proteins, viz., major ampullate spidroin1 (MaSp1) and major ampullate spidroin 2 (MaSp2).6−8 These two proteins can be described as highly repetitive block copolymers with alternating polyalanine (poly-A), typically 6 and 7 residues long, and Gly-rich blocks. Spider dragline silk fiber has been represented as a two-phase material comprising crystalline and amorphous regions, but this representation seems to be oversimplistic.9−16 Among many analytical methods,5,10 solid-state NMR alone gives the information about the structure and dynamics in an amino acid-specific sequence of the spider silk in the solid state and therefore can be used to construct the detailed molecular-level picture. 17,18 Many NMR researchers have studied the conformation of the silk polymer chains by using conformation-dependent NMR chemical shifts of amino acid residues19−37 coupled with selective stable-isotope labeling. There have been some controversies about the conformation of the Gly residues, i.e., 31-helix,21,23,28−31,38−40 random coil,19,20,41−43 © XXXX American Chemical Society

Received: December 11, 2017 Revised: January 16, 2018

A

DOI: 10.1021/acs.macromol.7b02627 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

previously,54 and a comparison was made between NCF and RSP in both the dry and hydrated states.

reported, there have been no investigations so far on the detailed packing structure of the poly-A regions. We studied previously46 the packing arrangements of (Ala)n (n = 3, 4, 5, 6, 7, 8, and 12) with antiparallel β-sheet structure, (AP-β-(Ala)n) as the simple model for the crystalline domain of spider dragline silks and wild silkworm silks by solid-state NMR and X-ray powder diffraction. Two different packing arrangements depending on the number of Ala residue could be discerned, viz., a rectangular arrangement for (Ala)6 and shorter poly-A chains and a staggered arrangement for (Ala)7 and longer poly-A chains.46,47 In addition, using solid-state NMR methods, we determined the packing arrangements of a wild silkworm, Samia cynthia ricini silk fibroin, which had a similar primary structure as MaSp1, i.e., block copolymers with alternating poly-A and Gly-rich blocks.48−50 The main difference between the silk fibroin and the spider silk was a longer poly-A sequence in the former silk fibroin, i.e., (Ala)12−13, relative to the spider silk. The poly-A sequences in S. c. ricini silk fiber were packed in the staggered arrangement. The atomic-level structure was determined using Cambridge Serial Total Energy Packing program and the NMR chemical shift calculation with Gauge Including Projector Augmented Wave method.45 In addition, the change in the packing pattern from rectangular to staggered arrangements was also observed for (Ala)6 by heating the sample at 200 °C for 4 h, which could also be monitored by molecular dynamics (MD) simulation at 200 °C.51 In this paper, we aim to reveal the packing arrangement of poly-A region with antiparallel β-sheet structure in N. clavata dragline silk fiber (NCF) in the dry and hydrated states. This spider is often known as the Joro spider and is native to East Asia (Japan, China, Korea, and Taiwan).52 The primary structure of the dragline silk has been partially reported.53 Therefore, we first observed the 13C solution NMR spectrum of NCF and compared it with that of recombinant spider silk protein (RSP) reported previously.54 For the structural and dynamical characterizations of NCF in both the dry and hydrated states, the combination of three solid-state NMR methods (13C refocused INEPT, DD/MAS, and CP/MAS) was used. 55,56 The refocused INEPT observes the mobile components of the hydrated NCF with fast isotropic motion (>105 Hz). In contrast, 13C CP/MAS NMR selectively observes the immobile components of NCF or those with very slow motion (