ABSTRACT:We have previously suggested that crystalline Bombyx mori silk in silk 16 II form (the silk structure after spinning) is not a simple antiparallel β-sheet but is 17 intrinsically heterogeneous. Using the peptide (AG) 15 36 and dried under mild conditions) has been shown to possess a 37 repeated type II β-turn structure. 7−9 On the other hand, the 38 precise intermolecular packing in the Silk II form (representing 39 the core of the spun silk fiber) has not yet been determined. 40 Using X-ray fiber diffraction of the crystalline region, the 41 structure of Silk II was first characterized by Marsh, Corey, and 42 Pauling 10 as a regular array of antiparallel β-sheets: this 43 structure remains the classic image of β-sheet silk. We call this 44 cxs00 | ACSJCA | JCA10.0.1465/W Unicode | research.3f (R3.6.i5 HF05:4232 | 2.0 alpha 39) 2014/10/10 09:17:00 | PROD-JCA1 | rq_3109040 | 12/15/2014 13:54:33 | 9 | JCA-DEFAULT 65 using a small (Ala-Gly) 15 peptide as the model. The alternating 66 copolypeptide (Ala-Gly) n has been generally accepted as a good 67 model of the crystalline region, NMR spectra of (AG) n 68 correspond closely to those obtained using the crystalline 69 fraction of native silk II fibers, 7−16 and the torsion angles of the 70 straight backbone chains correspond to the typical angles of an 71 antiparallel β-sheet. 17 In previous 13 C solid state NMR studies 72 of (AG) n , the 13 Cβ signal of the Ala residues has been reported 73 to consist of three peaks. 15,16 The high-field peak was assigned 74 to a distorted β-turn/random coil, while the other two peaks 75 were assigned to antiparallel β-sheet structures with different 76 intermolecular arrangements. The key challenge lies in the ability to discern and resolve the 92 two kinds of antiparallel β-sheet chains with different 93 intermolecular packing arrangements, as detected here and in 94 the earlier 13 C CP/MAS NMR study. 15,16 We therefore carried 95 out a search of packing arrangements, guided by crystallo-96 graphic and NMR data; refined the resulting structures; and 97 tested them against experimental data. The peptide (AG) n 98 crystallizes in space group P2 1 , a rectangular unit cell with the 99 parameters a = 9.38 Å, b = 9.49 Å, and c = 6.98 Å. The Marsh 100 model places the molecular axis along b but is otherwise very 101 similar: a = 9.40 Å, b = 6.97 Å, and c = 9.20 Å. In order to 102 generate two kinds of β-sheet models with different 103 intermolecular arrangements, we had the idea to calculate 104 atomic coordinates for the chains, setting either c or b along the 105 molecular axis. For each of these two models, energy 106 optimization was performed. 9 1 H, 13 C, and 15 N chemical shifts 107 were then predicted for the two antiparallel β-sheet structures 108 using the GIPAW method. 23

Samia cynthia ricini is one of the wild silkworms and its silk fibroin (SF) consists of alternatively repeating poly-l-alanine (PLA) sequences as crystalline domain and glycine-rich sequences as noncrystalline domain; the structure is similar to those of spider silk and other wild silkworm silks. In this paper, we proposed a new staggered model for the packing arrangement of the PLA sequence through the use of the Cambridge Serial Total Energy Package program and a comparison of the observed and calculated chemical shifts of the PLA sequence with the Gauge Including Projector Augmented Wave method. The new model was supported by the interatomic distance information from the cross peaks of Ala Cβ dipolar-assisted rotational resonance (DARR) spectrum of the PLA sequences in S. c. ricini SF fiber. In addition, three C NMR peaks observed in the β-sheet region were assigned to the carbons with different environments in the same model, but not assigned to different β-sheet structures.

Silk fibroin fiber is a well-known textile, but is also used as biomaterial that is of interest for use in a variety of applications, usually in a hydrated state. Thus, the determination of the hydrated silk fibroin structure is important for understanding the function and in designing novel biomaterials. In this work, 13 C dipolar decoupling/magic angle spinning NMR was used to determine the local conformation of [3-13 C]Ala-, [3-13 C]Ser-, and [3-13 C]Tyr -Bombyx mori silk fibroin in a hydrated state. Ser residues are present predominantly in the crystalline domains, Tyr predominantly in the noncrystalline domains and Ala residues in both domains. The fraction of β-sheet and two random coil distributions with fast and slow chain dynamics could be determined for all these residues by 13 C conformation-dependent chemical shift. The fraction of β-sheet of these residues in the fiber and the crystalline fraction did not change significantly before and after hydration. The fraction of random coil conformations with fast motion in total random coil fraction of the hydrated fiber was 25%, 22%, and 11% for Ala, Ser, and Tyr residues, respectively. Thus, Tyr residues tend to hydrate relatively little among these residues. This information is the first detailed study of the effects of hydration on site-specific crystalline and noncrystalline domains of silk.

Polyalanine (polyA) sequences are well known as the simplest sequence that naturally forms anti-parallel β-sheets and constitute a key element in the structure of spider and wild silkworm silk fibers. We have carried out a systematic analysis of the packing of anti-parallel β-sheets for (Ala), n = 5, 6, 7 and 12, using primarily C solid-state NMR and MD simulation. HFIP and TFA are frequently used as the dope solvents for recombinant silks, and polyA was solidified from both HFIP and TFA solutions by drying. An analysis of Ala Cβ peaks in theC CP/MAS NMR spectra indicated that polyA from HFIP was mainly rectangular but polyA from TFA was mainly staggered. The transition from the rectangular to the staggered arrangement in (Ala) was observed for the first time from the change in the Ala Cβ peak through heat treatment at 200 °C for 4 h. The removal of the bound water was confirmed by thermal analysis. This transition could be reproduced by MD simulation of (Ala) molecules at 200 °C after removal of the bound water molecules. In this way, the origin of the stability of the different packing arrangements of polyA was clarified.