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

The accurate (1)H positions of alanine tripeptide, A(3), with anti-parallel and parallel β-sheet structures could be determined by highly resolved (1)H DQMAS solid-state NMR spectra and (1)H chemical shift calculation with gauge-including projector augmented wave calculations.